1 Ph.D.-12 phage display library (X12) 12 ~1.0e9 ~1.0e13 New England BioLabs, Beverly, MA, USA Completely random NNK Linear The randomized peptide sequences in the library are expressed between the leader sequence and the mature N-terminus of the minor coat protein pIII, resulting in an average valency of 5 displayed peptides per virion. For the linear Ph.D.-12 libraries, the first position of the mature displayed fusion protein is the first randomized position. Each displayed peptide is followed by a short spacer (Gly-Gly-Gly-Ser) and then the wild-type pIII sequence. The display vector used for the library is a derivative of wild-type M13 phage-simple propagation of the library as phage rather than plasmid eliminates the need for antibiotic selection and a separate helper phage superinfection step, saving time and effort. Extensive sequencing of the library has revealed a wide diversity of side chains at each position with no obvious positional biases. 2 Ph.D.-7 phage display library (X7) 7 ~1.0e9 ~1.0e13 New England BioLabs, Beverly, MA, USA Completely random NNK Linear The randomized peptide sequences in the library are expressed between the leader sequence and the mature N-terminus of the minor coat protein pIII, resulting in an average valency of 5 displayed peptides per virion. For the linear Ph.D.-7 libraries, the first position of the mature displayed fusion protein is the first randomized position. Each displayed peptide is followed by a short spacer (Gly-Gly-Gly-Ser) and then the wild-type pIII sequence. The display vector used for the library is a derivative of wild-type M13 phage-simple propagation of the library as phage rather than plasmid eliminates the need for antibiotic selection and a separate helper phage superinfection step, saving time and effort. Extensive sequencing of the library has revealed a wide diversity of side chains at each position with no obvious positional biases. 3 Ph.D.-C7C phage display library (CX7C) 7 ~1.0e9 ~1.0e13 New England BioLabs, Beverly, MA, USA Completely random NNK Circular The randomized peptide sequences in the library are expressed between the leader sequence and the mature N-terminus of the minor coat protein pIII, resulting in an average valency of 5 displayed peptides per virion. For the Ph.D.-C7C disulfide-constrained library, the randomized sequence is preceded by Ala-Cys at the N-terminus. Each displayed peptide is followed by a short spacer (Gly-Gly-Gly-Ser) and then the wild-type pIII sequence. The display vector used for the library is a derivative of wild-type M13 phage-simple propagation of the library as phage rather than plasmid eliminates the need for antibiotic selection and a separate helper phage superinfection step, saving time and effort. Extensive sequencing of the library has revealed a wide diversity of side chains at each position with no obvious positional biases. 4 f3-6mer phage display library (X6) 6 2.0e8 G. Smith (University of Missouri, Columbia, MO) Completely random NNK Linear Library f3-6mer (GenBank Accession AF246446) is based on vector fUSE5, which displys its guest peptides on all five copies of the minor coat protein pIII at one tip of the virion. The library is also known as fUSE/6mer. 5 f3-15mer phage display library (X15) 15 2.5e8 G. Smith (University of Missouri, Columbia, MO) Completely random NNK Linear Library f3-15mer (GenBank Accession AF246445) is based on vector fUSE5, which displys its guest peptides on all five copies of the minor coat protein pIII at one tip of the virion. The library is also known as fUSE/15mer. 6 f88-15mer phage display library (X15) 15 2.0e9 G. Smith (University of Missouri, Columbia, MO) Completely random NNK Linear Library f88-15mer (GenBank Accession AF246448) is based on vector f88-4, which displays its guest peptides on 100 to 300 copies of the major coat protein pVIII. The library is also known as f88/15mer. 7 ACX7CG phage display library 7 2.3e8 Christian Heinis (Institute of Chemical Sciences and Engineering, Switzerland) Completely random NNK Circular The peptides containing seven random amino acids flanked on each side by cysteines were displayed as fusions with the minor coat protein pIII on filamentous phage. 8 f88-Cys1 phage display library (X5CXCX5) 13 2.8e9 G. Smith (University of Missouri, Columbia, MO) Semi-random NNK Circular Library f88-Cys1 (GenBank Accession AF246450) is based on vector f88-4, which displays its guest peptides on 100 to 300 copies of the major coat protein pVIII. The library is also known as Cys1. 9 f88-Cys2 phage display library (X5CX2CX5) 14 5.5e7 G. Smith (University of Missouri, Columbia, MO) Semi-random NNK Circular Library f88-Cys2 (GenBank Accession AF246451) is based on vector f88-4, which displays its guest peptides on 100 to 300 copies of the major coat protein pVIII. The library is also known as Cys2. 10 f88-Cys3 phage display library (X5CX3CX4) 14 5.2e7 G. Smith (University of Missouri, Columbia, MO) Semi-random NNK Circular Library f88-Cys3 (GenBank Accession AF246452) is based on vector f88-4, which displays its guest peptides on 100 to 300 copies of the major coat protein pVIII. The library is also known as Cys3. 11 f88-Cys4 phage display library (X4CX4CX5) 15 1.7e8 G. Smith (University of Missouri, Columbia, MO) Semi-random NNK Circular Library f88-Cys4 (GenBank Accession AF246453) is based on vector f88-4, which displays its guest peptides on 100 to 300 copies of the major coat protein pVIII. The library is also known as Cys4. 12 AXCX5CXG phage display library 9 1.05e7 Christian Heinis (Institute of Chemical Sciences and Engineering, Switzerland) Semi-random NNK Circular The peptides containing five random amino acids flanked on each side by cysteines were displayed as fusions with the minor coat protein pIII on filamentous phage. 13 f88-Cys6 phage display library (X4CX6CX4) 16 2.7e8 G. Smith (University of Missouri, Columbia, MO) Semi-random NNK Circular Library f88-Cys6 (GenBank Accession AF246454) is based on vector f88-4, which displays its guest peptides on 100 to 300 copies of the major coat protein pVIII. 14 f88-LX6 phage display library (XCX6CX) 10 1.0e10 J. K. Scott Semi-random NNM Circular f88-LX6 (GenBank accession number AF246456) were constructed in vector f88-4, which displays its guest peptides on 100 to 300 copies of the major coat protein pVIII. Met and Trp are absent from this library. 15 f8-8mer phage display library (X8) 8 2.0e9 V. A. Petrenko and G. P. Smith Completely random NNK Linear Library f8-8mer (GenBank accession number AF246447) was constructed in vector f8-1, which displays its guest peptides on all 3,900 copies of pVIII. In fact, codons of X8 is Gnk-(nnk)6-nnG. 16 CL6 phage display library (CX6C) 6 9.0e8 1.0e13 L. Mazzucchelli and J. B. Burritt Completely random NNK Linear CL6 was constructed using a vector M13KBstX, which has kanamycin resistance. 17 CL10 phage display library (CX10C) 10 1.04e19 1.0e13 L. Mazzucchelli and J. B. Burritt Completely random NNK Circular CL10 was constructed using a vector M13KBstX, which has kanamycin resistance. 18 LL9 phage display library (X9) 9 3.66e9 L. Mazzucchelli and J. B. Burritt Completely random Linear 19 J404 phage display library (X9) 9 L. Mazzucchelli and J. B. Burritt Completely random NNK Linear J404 is a linear random nonapeptide M13 phage display library with kanamycin resistance. 20 X6 fAFF1 phage display library 6 3.0e8 W. Dower (Affymax) Completely random NNK Linear The library is based on a filamentous bacteriophage affinity vector, fAFF1, which was constructed from the tetracycline resistance-transducing vector fd-tet. Random peptides were express on protein III. Coding scheme: NNK, where N represents equimolar A, C, G, or T and K is equimolar G or T. NH2-XXXXXXGG=TVESC, amino acids after the equals sign belong to the native gene III protein. 21 X12 fAFF1 phage display library 12 3.0e8 W. Dower (Affymax) Completely random NNK Linear The library is based on a filamentous bacteriophage affinity vector, fAFF1, which was constructed from the tetracycline resistance-transducing vector fd-tet. Random peptides were express on protein III. 22 X20 fAFF1 phage display library 20 4.0e8 W. Dower (Affymax) Completely random NNK Linear The library is based on a filamentous bacteriophage affinity vector, fAFF1, which was constructed from the tetracycline resistance-transducing vector fd-tet. Random peptides were express on protein III. 23 ON1203 phage display library (CX8C) 8 2.4e9 Affymax Completely random Circular The gene for pVIII was chemically synthesized with the use of four overlapping oligonucleotides. The gene was inserted into the Nhe and Hind IlIl sites of the phagemid vector pBAD18, placing the expression under the control of the araB promoter (plasmid p8V2). A cloning site consisting of two noncomplementary Bst Xl sites was included at the 5' end of the gene for pVIII and was used to clone collections of degenerate oligonucleotides by the half-site primer approach as described. Library ON1203 was constructed in this vector. 24 X6 fAFF1-tether C phage display library 6 2.0e8 Marc Navre (Affymax) Completely random NNK Linear This library is based on fAFF1 vector, displaying random hexapeptides linked to pIII. 25 X8, X10 and X12 phage display library pool 8, 10, 12 Affymax Completely random Linear Libraries of random peptides were expressed at the N terminus of fd phage pIII protein or on the N terminus of the fd phage major coat protein pVIII. 26 pVIII-9aa phage display library (X9) 9 F. Felici (Istituto di Ricerche di Biologia Molecolare, Rome, Italy) Completely random NNN Linear The random nonapeptides were inserted into the amino terminus of the pVIII protein coat of bacteriophage fl. 27 pVIII-9aa.Cys phage display library (CX9C) 9 F. Felici and A. Luzzago (Istituto di Ricerche di Biologia Molecolare, Rome, Italy) Completely random NNN Circular Phages is this library contain peptide inserts of 9 random amino acid residues, flanked by two invariant cysteine residues, within the major M13 phage coat protein pVllI. The insert sequences are preceded by AEGEF and followed by GDPAK. 28 pVIII-9aa and pVIII-9aa.Cys phage display library pool 9 F. Felici (Istituto di Ricerche di Biologia Molecolare, Rome, Italy) Completely random NNN pVIII-9aa contained "unconstrained" nonapeptides inserted into the amino terminus of the pVIII coat protein of bacteriophage f1. pVIII-9aa.Cys contained nonapeptides "constrained" by a disulphide bond formed by an additional cysteine at each end of the peptide. Phagemids were propagated using the related helper phage M13KO7 (Promega, Madison, USA). 29 f88-4/15mer and f88-4/Cys4 phage display library pool 14, 15 G. Smith (University of Missouri, Columbia, MO) Semi-random NNK Two phagemid libraries expressing random peptides fused to the N terminus of the pVIII coat protein of M13 phage were mixed in equal proportions. One library (f88-4/15mer) contained linear 15-mer peptides whereas the second (f88-4/Cys4) contained 14-mer peptides with conformation constrained by the presence of cysteine residues within the peptide at positions 5 and 10. 30 X20 fUSE5 phage display library 20 Michael Foley Completely random Linear 31 CX7C fUSE5 phage display library 7 Martin Trepel (University of Freiburg Medical Center, Germany) Completely random NNK Circular The fUSE5 virions were obtained from Dr. G. Smith (University of Missouri, Columbia, MO). 32 CX4C and X6 phage display library pool 4, 6 John C. Reed Completely random 33 CX7C T7 phage display library 7 G. Froman (Department of Medical Microbiology, Uppsala University, Sweden) Completely random NNK Circular 34 X10 phage display library 10 7.2e10 Yipeng Qi Completely random NNK Linear 35 X7+CX7C+CX10C+CX3CX3CX3C phage display library pool 7, 10, 11 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Semi-random All libraries are based on vector fUSE5. 36 X10 phage display library 10 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Completely random Linear X10 is based on vector fUSE5. 37 X11 phage display library 11 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Completely random Linear X11 is based on vector fUSE5. 38 CX6C phage display library 6 Corvas (Gent, Belgium) Completely random Circular 39 X15+XCX8CX+X9+CX10C phage display library pool 9, 10, 12, 15 Claude Granier Semi-random Two M13 phage display library pool were obtained that express either linear 15-mer random peptides (X15) or constrained 12-mer random peptides (XCX8CX) at the surface of the pVIII protein. Two additional phage display library pool, expressing either linear random nonapeptides (X9) or constrained random dodecapeptides (CX10) at the surface of the pIII protein, were obtained. 40 f3-15mer and f88-15mer phage display library pool 15 G. Smith (University of Missouri, Columbia, MO) Completely random NNK Linear Library f3-6mer (GenBank Accession AF246445) is based on vector fUSE5, which displys its guest peptides on all five copies of the minor coat protein pIII at one tip of the virion. The library is also known as fUSE/15mer. Library f88-15mer (GenBank Accession AF246448) is based on vector f88-4, which displays its guest peptides on 100 to 300 copies of the major coat protein pVIII. The library is also known as f88/15mer. 41 J404-3 phage display library (X9) 9 J. B. Burritt Completely random Linear 42 X6 phage display library 6 8.0e8 J. K. Scott (Simon Fraser University, Burnaby, Canada) Completely random NNK Linear The library is based on vector f88-4 (GenBank Accession AF218363). 43 X15 phage display library 15 1.3e9 J. K. Scott (Simon Fraser University, Burnaby, Canada) Completely random NNK Linear The library is based on vector f88-4 (GenBank Accession AF218363). 44 X30 phage display library 30 2.5e8 J. K. Scott (Simon Fraser University, Burnaby, Canada) Completely random NNK Linear The library is based on vector f88-4 (GenBank Accession AF218363). 45 X8CX8 phage display library 17 2.5e8 J. K. Scott (Simon Fraser University, Burnaby, Canada) Semi-random NNK Linear The library is based on vector f88-4 (GenBank Accession AF218363). 46 X15CX phage display library 17 1.2e8 J. K. Scott (Simon Fraser University, Burnaby, Canada) Semi-random NNK Linear The library is based on vector f88-4 (GenBank Accession AF218363). 47 XCX15 phage display library 17 1.0e9 J. K. Scott (Simon Fraser University, Burnaby, Canada) Semi-random NNK Linear The library is based on vector f88-4 (GenBank Accession AF218363). 48 LX-4 phage display library (XCX4CX) 8 2.2e8 J. K. Scott (Simon Fraser University, Burnaby, Canada) Semi-random NNK Circular The library is based on vector f88-4 (GenBank Accession AF218363). 49 LX-6b phage display library (XCX6CX) 10 1.5e8 J. K. Scott (Simon Fraser University, Burnaby, Canada) Semi-random NNK Circular The library is based on vector f88-4 (GenBank Accession AF218363). 50 LX-8 phage display library (XCX8CX) 12 1.5e9 J. K. Scott (Simon Fraser University, Burnaby, Canada) Semi-random NNK Circular The library is based on vector f88-4 (GenBank Accession AF218363). 51 Alpha-CT phage display library (XCCX3CX5C) 13 5.0e8 J. K. Scott (Simon Fraser University, Burnaby, Canada) Semi-random NNK Circular The library is based on vector f88-4 (GenBank Accession AF218363). 52 LX8 and X15CX phage display library pool 12, 17 J. K. Scott (Simon Fraser University, Burnaby, Canada) Semi-random NNK 53 X15 and X21 phage display library pool 15, 21 Anthony J. Conley Completely random NNK Linear 54 CX12C phage display library 12 Tel-Aviv University (TAU) Completely random Circular The library is based on the fth-1 expression vector in which random peptides are recombinantly fused to the NH2 terminus of a recombinant protein VIII of the fd filamentous bacteriophage. 55 f3-6mer and J404-3 phage display library pool 6, 9 Completely random NNK Linear Library f3-6mer (GenBank Accession AF246446) is based on vector fUSE5, which displys its guest peptides on all five copies of the minor coat protein pIII at one tip of the virion. The library is also known as fUSE/6mer. 56 CX6C phage display library 6 Igor Fisch Completely random Circular 57 X6 phage display library 6 Igor Fisch Completely random Linear 58 X4[RK]X3 phage display library 7 Richard L. Stevens Semi-random NNN Linear Phage display peptide library is tryptase specific. The genome of the Ff bacteriophage consists of the 11 genes designated gI to gXI. Although the protein (pIII) encoded by gIII is chymotrypsin-, thermolysin-, and subtilisin-susceptible, it is trypsin-resistant. Thus, phage display peptide libraries can give insight into the substrate specificities of certain proteases. 59 L100 phage display library (X10) 10 6.0e8 Matthew D. Scharff (Einstein College, New York) Completely random NNK Linear L100 is based on fUSE5. 60 CX9C phage display library 9 C. Richter King Completely random Circular The library was constructed to contain a variable 9-amino acid peptide flanked by cysteine residues inserted into the GeneIII protein of the phage fUSE5. 61 S-(X)5-FRW-(X)4 phage display library 9 1.0e5 Jarl E. S. Wikberg Semi-random Linear A small library was constructed to test this phage display system using a partially randomized oligonucleotide (MS-5pcr) that kept the MSH-core sequence Phe7-Arg8-Trp9, which is believed to be most essential for receptor binding, to direct phage binding toward the receptors. The Ser at the junction with the pelB leader sequence was also maintained to reduce the amount of sequences that may not be processed by the leader peptidase. 62 Cys6 phage display library (CX6C) 6 2.2e7 T. E. Michaelsen Completely random Circular The library present peptides of six random amino acids constrained by flanking cysteines were created by insertion of double-stranded oligonucleotides between the Sfi1 site of the vector fUSE5, providing the gene 3 protein (g3p) final amino acid sequences NH2-ADGACX6CGAA-g3p for the hexamer library (cys6), where X stands for any amino acid. 63 Cys9 phage display library (CX9C) 9 5.5e7 T. E. Michaelsen Completely random Circular The library present peptides of nine random amino acids constrained by flanking cysteines were created by insertion of double-stranded oligonucleotides between the Sfi1 site of the vector fUSE5, providing the gene 3 protein (g3p) final amino acid sequences ADGACX9CGAA-g3p for the nanomer library (cys9), where X stands for any amino acid. 64 X9 phage display library 9 Heimo Breiteneder Completely random Linear Random peptides are displayed on filamentous phage as fusion to the NH2 terminus of the major coat protein pVIII. 65 X15 phage display library 15 Michael Jaye Completely random NNK Linear A fusion phage display library was constructed in the filamentous bacteriophage fuse 5B, a vector derived from fd filamentous phage. The fuse5B vector was constructed from the vector fuse5 by removal of an existing BstXI site in fuse5 followed by replacement of the SfiI cloning sites with BstXI cloning sites. 66 GX6G phage display library 6 2.5e9 Zoller and Smith Completely random NNK Linear 67 X6PPIP phage display library 6 2.0e9 Zoller and Smith Completely random NNK Linear 68 RSLRPLX6 phage display library 6 5.8e8 Zoller and Smith Completely random NNK Linear 69 PPPYPPX6 phage display library 6 3.1e8 Zoller and Smith Completely random NNK Linear 70 RKRSHRXXPPPXXXVQ phage display library 5 Christian Freund Semi-random Linear Sequences of the focused library RKRSHRXXPPPXXXVQ were cloned into PC89. PC89 was a gift from Gianni Cesareni(Dipartimento di Biologia, Universita di Roma). 71 X9 phage display library 9 Gianni Cesareni(Dipartimento di Biologia, Universita di Roma) Completely random Linear 72 X10 phage display library 10 1.0e8 Daniel Baty Completely random NNK Linear 73 X12 phage display library 12 Peptide Door, Japan Completely random Linear 74 X6 phage display library 6 1.0e7 5.4e8 Michael N. Margolies Completely random Linear A bacteriophage-displayed library contains randomized mutations at H chain residues 30-35 of the anti-digoxin antibody 26-10 Fab. 75 AA-xxxxx-AA phage display library 5 2.0e6 James A. Wells Completely random Linear 76 AA-xxRx(K/R)Rx-AA phage display library 7 James A. Wells Semi-random Linear 77 AA-xxRxPRx-AA phage display library 7 James A. Wells Semi-random Linear 78 X10 phage display library 10 Matthew D. Scharff Completely random NNK Linear The library is based on fUSE5 79 L200 phage display library 19 Matthew D. Scharff Semi-random Linear The L200 library was created by inserting a modified version of the PA1 motif between the SfiI sites of the vector fUSE5, located at the amino-terminal part of the pIII coat protein giving the final amino acid sequence H2N-ADVA X6 TPXW[M/L][M/L] X6 AAG-g3p. 80 CX(7-10)C and X(9-10) phage display library pool 7-10 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Completely random 81 Three CX12C phage display library pool 12 Jonathan M. Gershoni Completely random Circular Each library represented 1e9 - 5e9 recombinant random 12mer peptides flanked by constant cysteine residues so as to constrain a looped structure at the NH2 terminus of the major coat protein, pVIII, of the folamentous bacteriophage fd. Three 12mer cysteine-constrained peptide libraries were constructed: one in ftac88 and the other two in fth1. 82 CX12C phage display library 12 Jonathan M. Gershoni Completely random Circular 83 X6PX6 and X6YX6 phage display library pool 12 Mark W. Grinstaff Semi-random NNK Linear 84 CX6C phage display library 6 6.4e7 1.1e9 Indraneel Ghosh Completely random NNS Circular The six-residue disulfide-constrained cyclic peptide library was constructed N-terminal to a peptide linker to the gene III fusion protein encoded by the phagemid vector pCANTAB-5E (Amersham Biosciences, Princeton, NJ, USA). A gene encoding a peptide linker and containing an internal PstI restriction site had been previously cloned into pCANTAB-5E between SfiI and NotI restriction sites in our laboratory to produce pCANTAB-Fos. 85 CX7C T7 phage display library 7 1.28e9 3.3e7 University of Tartu, Tartu, Estonia Completely random NNK Circular This library was constructed using the T7 select-415 kit from Novagen with NNK codon. Target peptides were expressed as a fusion to the C-terminus of the 10B capsid protein and were displayed on the virion surface where they were accessible for interaction with other proteins or ligands. The displayed peptide was situated between cysteine residues and therefore, formation of a disulphide bridge would join the ends of the heptapeptide. The fusion polypeptide was present in 415 copies on each phage particle. 86 X12 phage display library 12 Jonathan M. Gershoni Completely random Linear 87 Ph.D.-12, Ph.D.-7 and Ph.D.-C7C phage display library pool 7, 12 New England BioLabs, Beverly, MA, USA Completely random NNK 88 X5-Fixed-X5 phage display library 10 James R. Burke Completely random Linear An assortment of libraries representing different fixed residue and other structural constraints will provide the greatest likelihood of deriving high-affinity peptides specific for a given target. 89 fUSE5-based CX7C phage display library 7 2e8 1e12 Renata Pasqualini (University of Texas, M. D. Anderson Cancer Center, Houston, TX) Completely random Circular 90 Type 8 phage display library (X8) 8 Angela M. Belcher Completely random NNM Linear The M13KE phage vector was modified by making a cloning site for pVIII display. A Pst I restriction site was made by mutating T to A at position 1372, a BamH I site was made by mutating C to G at positon 1381, and the Pst I site at position 6246 was deleted by mutating T to A at position 6250. The site-directed mutagenesis was done using overlap extension PCR. A dsDNA library was then prepared and cloned into the resulting modified phage vector, named M13SK, using Pst I and BamH I. To obtain the dsDNA library, partial library duplexes were formed by annealing of extension primer (5\'-GATGCTGTCTTTCGCTGCAG-3\') with oligonucleotides (3\'-ACGACAGAAAGCGACGTCnm(nnm)6nnCCTAGGAACATC ATC-5\', where n=A, T, C, or G and m=A or C). The partial library duplexes were incubated with Klenow fragment (3\'→'5\' exo-) (10 U/μL) and dNTP at 37 °C for 30 min. The Klenow fragment was inactivated by heating (75 °C for 20 min), and the mixture was digested with Pst I and BamH I. The digested DNA was gel purified (2-40% TBE polyacrylamide gel), ligated into M13SK, and transfected to XL1-Blue Electroporation Competent Cells using a MicroPulser (Biorad). The library was titered according to manufacturer directions and sequenced (MIT Biopolymers Laboratory) before amplification. 91 pSKAN8-HyA phage display library 8 MoBiTec GmbH (Gottingen, Germany) Completely random Circular The variable region is a 8-aa (pSKAN8-HyA) extended peptide held between two disulfide bridges at the exposed tip of the human pancreatic secretory trypsin inhibitor (PSTI). A phagemid pSKAN8 was created which contains a fusion between the PSTI and M13 pIII protein-coding genes. 92 X12 phage display library 12 Antonio Verdoliva Completely random Linear The library was based on pC89. 93 X12 phage display library 12 Nicholas M. Boulis Completely random Linear 94 CX7C phage display library 7 MoBiTec GmbH (Gottingen, Germany) Completely random Circular 95 X4YX4 phage display library 8 T.S. Pillay Semi-random Linear The library was constructed by subcloning oligonucleotides encoding a random nonapeptide sequence into the 10B gene of the icosahedral T7 bacteriophage, using the T7 Select 415-1b cloning kit (Novagen). The cloned oligonucleotide, 5'-(NNB)4-TAT-(NNB)4-3', of the synthetic construct contained a conserved tyrosine (codon TAT) residue critical for phosphorylation at the center. 96 X6PPX(Y/F)X6 phage display library 13 Bernard Weisblum Semi-random Linear phage display library was based on insertion of a random DNA sequence, as indicated, in a multiple cloning site at the N-terminus of phage M13 protein pIII 97 CX9C and CX15C phage display library pool 9, 15 M. A. Persson Completely random Circular 98 X12 phage display library 12 Yuan Wang and You-hua Xie Completely random Linear The gene for the pVIII protein of phage M13 was amplified by PCR with VCSM13 (Stratagene, La Jolla, CA) as the template and inserted into pCANTAB5E (Amersham Pharmacia, Uppsala, Sweden), replacing the original gene III. The new vector, designated pFuse8, was used as parental vector for pVIII-based phage display. 99 CDX3KPCALLRYX10 phage display library 13 Igor Fisch Completely random Circular 100 LX-8, X15, X8CX8, X30 phage display library pool 12, 15, 17, 30 J. K. Scott (Simon Fraser University, Burnaby, Canada) Semi-random This is a mixture phage display library (LibraryID: 43-45,50). 101 CX7C fUSE5 phage display library 7 Erkki Ruoslahti Completely random Circular 102 CX10C phage display library 10 6.2e8 Xiaomin Fan Completely random Circular 103 pAFF/MBP vector-based phage display library 8 3.0e9 1.0e12 A. Gonzalez-Techeraa Completely random Circular This library, with the general sequence ASGSACX8CGP6, expresses cyclic 8 mer random peptides flanked by two cysteines and fused to the phage coat protein pIII. 104 pComb3 X15 phage display library 15 1e10 H. Pannekoek, Amsterdam Medical Centre, Amsterdam, The Netherlands Completely random Linear 105 pComb8 CX15C phage display library 15 1e10 H. Pannekoek, Amsterdam Medical Centre, Amsterdam, The Netherlands Completely random Circular 106 X13 phage libray 13 1e8 1e12 Dyax Corp Completely random Linear This library displays 13 amino acids of randomized sequence equimolar at each position for all amino acids except cysteine. 107 CX7C T7 phage display library 7 5e8 1e9 Hai-yan Hong Completely random NNK Circular The phage library displayed CX7C (C, cysteine; X, any amino acid residue) random peptides, which was built in the T7 415-1b vector (Novagen) between EcoRI and HindIII sites. X residues are encoded by NNK. 108 X6 phage display library 6 2.0e8 4.5e10 M Trepel Completely random NNK Linear 109 CX7C phage display library 7 M Trepel Completely random NNB Circular 110 Tat-based pCANTAB-5E library library 5 Haruhiko Kamada Semi-random Linear The library has a framework as GX5QX3PX2, randomizing ten amino acids within the Tat transduction domain. 111 CX7C T7 phage display library 7 1.0e8 1e9 Novagen Completely random NNK Circular This library is based on T7 415-1b phage vector. 112 X9 phage display library 9 1.0e10 Carla Lo Passo Completely random Linear 113 X12 phage display library 12 1.0e10 Carla Lo Passo Completely random Linear 114 X7-LXXLL-X7 M13 phage display library 16 Niharika B. Mettu Semi-random Linear 115 SS-X16-S M13 phage display library 16 D. J. Kenan (Duke University) Completely random Linear 116 X8 and CX8C phage display library pool 8 Anthony N. Hodder Completely random The pool consists two eight-residue libraries displaying peptides on M13 gene VIII coat protein. One is a linear library with all 8 residues randomized. Another is a cyclic library with all 8 residues randomized in the context of a pair of fixed cysteine residues. In the cyclic library, the spacing of the cysteine residues varied from three to six residues. The randomization of residues for both libraries was achieved using Kunkel mutagenesis, employing degenerate oligonucleotides in which each randomized residue was encoded by an NNS codon. Equal aliquots of each linear and cyclic sublibrary were combined, giving rise to the 8-residue pool for panning. 117 X14 and CX14C phage display library pool 14 Anthony N. Hodder Completely random The pool consists two 14-residue libraries displaying peptides on M13 gene VIII coat protein. One is a linear library with all 14 residues randomized. Another is a cyclic library with all 14 residues randomized in the context of a pair of fixed cysteine residues. In the cyclic library, the spacing of the cysteine residues varied from three to six residues. The randomization of residues for both libraries was achieved using Kunkel mutagenesis, employing degenerate oligonucleotides in which each randomized residue was encoded by an NNS codon. Equal aliquots of each linear and cyclic sublibrary were combined, giving rise to the 14-residue pool for panning. 118 Display PHAGE system library (CX6C) 6 3e7 1e12 Display System Biotech (Vista, California) Completely random Circular 119 fUSE55-based X8 phage display library 8 1.1e8 George Georgiou Completely random NNK Linear The rondom sequence was inserted in gIII. 120 TN phage display library pool 9-12 Dyax Corp Semi-random Linear TN-pooled library is composed of TN-9-IV (3.2e9 independent transformants), TN10-X(2e9 independent transformants) and TN-12-I (1.4e9 independent transformants). 121 CX9C M13 phage display library 9 7.0e8 Kerry S. Kelleher Completely random Circular The CX9C library was constructed using the M13 filamentous bacteriophage peptide display cloning system from New England Biolabs. Expressing the peptide as a fusion with the M13 minor coat protein gene III presents as many as five copies of the peptide clustered on the surface of the bacteriophage. 122 CX9W1-9C M13 phage display library 9 Kerry S. Kelleher Semi-random Circular The CX9W1-9C library was designed to contain at least one tryptophan residue in every phage displayed peptide. This library was constructed using the M13 filamentous bacteriophage peptide display cloning system from New England Biolabs. Expressing the peptide as a fusion with the M13 minor coat protein gene III presents as many as five copies of the peptide clustered on the surface of the bacteriophage. 123 LX10 phage display library (CX10C) 10 Mark Paetzel Completely random Circular Random 10-mer peptide sequence constrained by two conserved cysteines was displayed on the pVIII of the f88 phage. 124 X6 T7 phage display library 6 1.5e8 Morley D. Hollenberg Completely random NNK Linear The sequence of synthetic degenerate oligonucleotides inserted in the coding region of T7 phage capsid protein (employing T7 Select 1-1 vector arms, T7 Select system, Novagen), encoding a random hexamer followed by (His)6 tag. 125 fUSE5-based X15 phage display library 15 Dorothee Herlyn Completely random Linear 126 ANL 22 phage display library 20 1e10 Brian K. Kay Completely random Linear The ANL 22 library displayed X20 peptides as N-terminal fusions to the protein product of gene III of bacteriophage M13 using SAM vector. 127 Cys6 phage display library 6 2.2e7 Inger Sandlie Completely random Circular The Cys6 library was based on the vector fUSE5, which supports phage fd protein III (pIII) peptide expression. 128 Cys9 phage display library 9 5e7 Inger Sandlie Completely random Circular The Cys9 library was based on the vector fUSE5, which supports phage fd protein III (pIII) peptide expression. 129 NNK11 phage display library 11 9e8 Inger Sandlie Completely random NNK Linear The NNK11 library was based on the vector fUSE5, which supports phage fd protein III (pIII) peptide expression. 130 Evo1 phage display library 6 3e7 Inger Sandlie Semi-random Circular The Evo1 library was based on the vector fUSE5, which supports phage fd protein III (pIII) peptide expression. The insert sequence is XXXCPWFQWPCXXX, where X denotes any residue. 131 Evo2 phage display library 6 3e7 Inger Sandlie Completely random Linear The Evo2 library was based on the vector fUSE5, which supports phage fd protein III (pIII) peptide expression. The insert sequence is XXXXXXWFQWPC, where X denotes any residue. 132 Evo3 phage display library 6 3e7 Inger Sandlie Completely random Linear The Evo3 library was based on the vector fUSE5, which supports phage fd protein III (pIII) peptide expression. The insert sequence is CPWFQWPCXXXXXX, where X denotes any residue. 133 CPEP-8 phage display library (X3CX8CX3) 14 2e9 John Lund Semi-random NNS Circular In the CPEP-8 library, peptides are displayed at the N-terminal of the pIII mature protein on the M13 bacteriophage surface. 134 fdMED1-based X6 phage display library 6 2e8 2e9 Completely random Linear 135 Y-X10-M phage display library 10 Ichio Shimada Completely random Linear This library displayed foreign peptides on the N terminus of the major coat protein (gVIII protein) of bacteriophage M13. Each foreign peptide has a 10-mer random sequence flanked by a Y residue (detectable residue at 280 nm by high-performance liquid chromatography) and a M residue (cyanogen bromide cleavage residue). 136 G-α phage display library (XXLXXXAX) 8 8.4e3 V.A. Petrenko Semi-random NNK Linear G-α-library is a library of phages carrying b-galactosidase-binding peptide ADTFAKSMQ at the N-terminus of the pVIII protein surrounded by random amino acids. 137 fdMED1-based CX6C M13 phage display library 6 5e8 1e12 Mehdi Houimel Completely random Circular 138 X9 T7 phage display library 9 1e8 Roberto Diaz Completely random Linear Random nonamer peptides are displayed on T7 phages as fusion proteins with the amino terminus of 10A capsid protein. 139 CX7-10C T7 phage display library 7-10 Novagen Completely random NNK Circular A mixture of 4 cyclic libraries CXnC was used which displayed 415 copies of the peptide on the capsid. Combined average diversity of the libraries was 1e8. The libraries were based on the T7Select 415-1b vector from Novagen. 140 X7 T7 phage display library 7 1e7 Mark R. Spaller Completely random NNK Linear The library was based on the T7 select 415-1 vector from Novagen. Eahg phage particle can displays 415 X7 peptides. 141 M13LP67-based X12 phage display library 12 2.1e8 Yuzhang Wu Completely random Linear 142 Ph.D.-12 and Ph.D.-C7C phage display library pool 7, 12 New England BioLabs, Beverly, MA, USA Completely random NNK 143 f8-9mer phage display library (X9) 9 2.0e9 V. A. Petrenko Completely random NNK Linear 144 CX10C P99 β-lactamase-fusion library 10 6.1e7 Girja S. Shukla Completely random NNK Circular The library was constructed at the amino-terminus of the Enterobacter cloacae P99 cephalosporinase molecule (P99 β-lactamase) with NNK-scheme of randomization. The β-lactamase enzyme acted as a reporter, saving time and resources otherwise required by the phage-ELISA of a typical phage display screening. 145 X12 P99 β-lactamase-fusion library 12 4.9e7 Girja S. Shukla Completely random Linear The library was constructed at the amino-terminus of the Enterobacter cloacae P99 cephalosporinase molecule (P99 β-lactamase) with NNK-scheme of randomization. The β-lactamase enzyme acted as a reporter, saving time and resources otherwise required by the phage-ELISA of a typical phage display screening. 146 Three pools of fUSE5 phage display library 7-12 Chun-Mei Li Completely random Random peptides were inserted to the minor capsid protein PIII of a filamentous bacteriophage fUSE5. Three pools of such libraries with peptides displayed on the surface of the phage particles were screened. Pool I was composed of four libraries of linear 7- to 11-aa peptides and pool II of three libraries of 9- to 12-aa peptides crosslinked via terminal cysteines, and pool III contained a mix of five libraries with both linear and crosslinked peptides, 8 to 10 aa long. 147 Min-23-R10 phage display library 10 2.8e8 Christelle Souriau Completely random NNK Linear The library was constructed by PCR using a set of degenerate primers to amplify Min-23 and replace residues 17-20 by 10 random residues ( LMRCKQDSDCLAGSVC(X)10FCG ). 148 YLK-(NNK)6 phage display library 6 4.6e7 Fujie Tanaka Completely random NNK Linear The library was appended to the N-terminus of the peptide (YKLLKELLAKLKWLLRKLXXXXXX, X = any of the natural 20 amino acids). 149 (NNK)6-YLK Library 6 4.7e7 Fujie Tanaka Completely random NNK Linear The library was appended to the C-terminus of the peptide (XXXXXXYKLLKELLAKLKWLLRKL, X = any of the natural 20 amino acids). 150 X12 T7 phage display library 12 9e10 Novagen Completely random Linear 151 CXnC phage display library pool (n = 3-12) 3-12 Kim D. Janda Completely random Circular 152 CX7C phage display library 7 4.5e9 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Completely random NNK Circular The library was constructed on fUSE5 vector. 153 X3CX7CX3 phage display library 13 5.0e8 Yuan Wang Semi-random Circular To construct the vector pFuse8, M13 gene 8 was amplified using the genomic DNA of VCSM13 phage as template. Random peptides fused with pVIII assume a form of X3CX7CX3, where X stands for any amino acids. Two cysteine residues frank the 7 random amino acids in the middle. 154 X10 phage display library 10 1.2e9 Alessandro Pini Completely random Linear The decapeptide library DIP3 expressed on the minor phage coat protein pIII of M13 bacteriophage was constructed modifying the vector pDN332. 155 Substrate phage display library based on a modified pH0508b phagemid 5 3.6e6 1.8e8 Sylvain M. Cloutier Completely random NNS Linear 156 X15 phage display library 15 Sparks A B Completely random Linear 157 XCX15 phage display library 16 4e10 Lori Bonnycastle(Simon Fraser University,Burnaby,BC,Canada) Semi-random Linear 158 (X)7LXXLL(X)7 library 16 1.5e8 Donald P. Mcdonnell Semi-random NNK Linear The library in the format of (X)7LXXLL(X)7 was constructed essentially with the M13 phage-based cloning vector mBAX. 159 fTC-LIB-N6 phage display library 6 Marc Navre Completely random NNK Linear 160 X9 phage display library 9 Al Jesaitis, Montana State University Completely random Linear 161 X15 phage display library 15 Nishi, T Completely random Linear 162 fUSE-His-N6 phage display library 6 Shuichi Ohkubo Completely random NNK Linear A random hexamer substrate phage display library with a (His)6 at the NH2-terminal of pIII protein of filamentous phage (fUSE5) has been constructed. 163 CTLA-4 (X3-RGD-X3) scaffold library 6 Hennie R. Hoogenboom Semi-random Linear The complementarity determining region 3 (CDR3) loop of the CTLA-4 extracellular domain was evaluated as a permissive site. Authors replaced the nine amino acid CDR3-like loop of CTLA-4 with the sequence XXX-RGD-XXX (where X represents any amino acid). 164 RRT-SH3 phage display library 6 Kalle Saksela Completely random Linear 165 CX8C phage display library 8 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Completely random NNK Circular The phage display peptide library is constructed in the vector fUSE5-phage. 166 CX10C phage display library 10 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Completely random NNK Circular The phage display peptide library is constructed in the vector fUSE5-phage. 167 CX3CX3CX3C phage display library 9 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Semi-random NNK Circular The phage display peptide library is constructed in the vector fUSE5-phage. 168 CX3CX4CX2C phage display library 9 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Semi-random NNK Circular The phage display peptide library is constructed in the vector fUSE5-phage. 169 PDL-10R phage display library 10 Christian, R. B. Semi-random NNK Linear PDL-10R was constructed in fUSE-5 vector (kindly provided by Dr. George Smith, University of Missouri, Columbia, MO). 170 PDL-10Y phage display library 9 Christian, R. B. Semi-random NNK Linear PDL-10Y was constructed in fUSE-5 vector (kindly provided by Dr. George Smith, University of Missouri, Columbia, MO). The library contained a tyrosine (TAT) at the third position of the insert sequence. 171 X2CX14CX2 phage display library 16 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Semi-random Circular 172 X2CX18 phage display library 20 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Semi-random Linear 173 X8 phage display library 8 Quanxi Li Completely random NNK Linear A random octapeptide library was prepared by cloning chemically-synthesized degenerated oligos into monovalent displayed phage antibody expressing vector, which is derived from pComh3H. 174 X15 phage display library 15 L. Jespers, Leuven, Belgium Completely random Linear 175 M13lib phage display library 9 Juergen Hammer Completely random NNS Linear MDstufferbb DNA was digested to completion with Sad and KpnI, and the stuffer was removed by gel electrophoresis to reduce any background. 176 HyC, HyB and HyA phage display library pool 6-8 R????ttgen P, Collins J Completely random NNK Circular The synthesis and characterisation of such phagemid-display banks is described here, in which the variable region is a 7-amino acid (aa) (pSKAN8-HyB/C) or 8-aa (pSKAN8-HyA) extended peptide held between two disulfide bridges at the exposed tip of the human pancreatic secretory trypsin inhibitor (PSTI). A phagemid pSKAN8 was created which contains a fusion between the PSTI and M13 pIII protein-coding genes. Cassettes containing the sequences (NNK)8 [HyA], (NNK)7 [HyB] or (NNK)6GTT [Hy-C] (where K = G or T) were used to randomize the aa coding region in the trypsin-inhibitory loop (aa 17 to 23) of PSTI. 177 X9 and CX9C phage display library pool 9 R. Cortese (IRBM, Pomezia, Italy) Completely random 178 M13lib X9 phage display library 9 2.4e7 Francesco Sinigaglia Completely random NNS Linear Random peptide inserts of nine amino acids flanked at each end by four glycine residues were incorporated into the pIII phage coat protein of M13. AEL=GGGGX9GGGG=VP. 179 X7 phage display library 7 Martin Trepel Completely random Linear 180 CX7C phage display library 7 Martin Trepel Completely random Linear 181 X18 phage display library 18 Martin Trepel Completely random Linear 182 Beta-sheet (BS) phage display library (X4CX6CX4) 16 Martin Trepel Semi-random Linear 183 N+5 class I phage display library 5 7.5e7 Richard J.Rickles Completely random NNM Linear The phage display library was prepared in bacteriophage M13. NH2-AE=XXXXXRPLPPLPPP=TVESCL, amino acids between the two equals signs does not belong to the native gene III protein. X denotes random residue, which is encoded by MNN (N = A, G, C,T; M=C, A). 184 C+5 class I phage display library 5 5.4e7 Richard J.Rickles Completely random NNM Linear The phage display library was prepared in bacteriophage M13. NH2-AE=RSLRPLPPLPXXXXX=TVESCL, amino acids between the two equals signs does not belong to the native gene III protein. X denotes random residue, which is encoded by MNN (N = A, G, C,T; M=C, A). 185 C+5 class II phage display library 5 2.2e7 Richard J.Rickles Completely random NNM Linear The phage display library was prepared in bacteriophage M13. NH2-AE=GAAPPLPPRXXXXX=TVESCL, amino acids between the two equals signs does not belong to the native gene III protein. X denotes random residue, which is encoded by MNN (N = A, G, C,T; M=C, A). 186 CX6C M13 phage display library 6 6.1e8 Lutz B. Giebel Completely random NNS Circular Peptides were displayed on the surface of filamentous phage M13 as fusions to the N-terminus of minor coat protein, pIII, using the expression vector M13East. This vector is a derivative of sequencing vector M13mp19. Coding scheme: NNS, where N = G, A, T, C and S = G, C. 187 CX5C M13 phage display library 5 2.4e8 Lutz B. Giebel Completely random NNS Circular Peptides were displayed on the surface of filamentous phage M13 as fusions to the N-terminus of minor coat protein, pIII, using the expression vector M13East. This vector is a derivative of sequencing vector M13mp19. Coding scheme: NNS, where N = G, A, T, C and S = G, C. 188 CX4C M13 phage display library 4 3.8e8 Lutz B. Giebel Completely random NNS Circular Peptides were displayed on the surface of filamentous phage M13 as fusions to the N-terminus of minor coat protein, pIII, using the expression vector M13East. This vector is a derivative of sequencing vector M13mp19. Coding scheme: NNS, where N = G, A, T, C and S = G, C. 189 X15 M13 phage display library 15 2e7 James J. Devlin (Cetus, Chiron) Completely random NNS Linear Peptides were displayed on the surface of filamentous phage M13 as fusions to the N-terminus of minor coat protein, pIII, using the expression vector M13LP67. 190 PDL-20 phage display library 20 1.5e8 Gary S. Gray Completely random NNB Linear PDL-20 library was prepared by cloning a degenerate oligo into the fUSE5 vector. The 20-mer random segment was fused to the pIII protein of bacteriophage fd. Coding scheme: NNB (the first N = 25% each G, A, T and C; the second N = 19%G, 19%C, 31%A, and 31%T; the B = 39%G, 39%C and 22%T). Characterization of the PDL-20 libray showed that all amino acids were present at the expected frequency and that there was no positional bias. 191 X11GX12 phage display library 21 A. Rail Castaio Semi-random Linear The library contained a random 22-amino acid sequence with a Gly residue at position 12, expressed at the mature NH2-terminus of the gene VIII protein of the M13 bacteriophage, just after the leader peptide. 192 CX9 phage display library 9 1e9 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Completely random Linear The library was constructed by inserting CX9 peptides (C: cysteine; X: any amino acid) into the pIII protein of the fUSE5 vector. The inclusion of one cysteine was intended to facilitate the selection of peptides containing cysteine pairs, should the binding activity studied require a cyclic structure. 193 8-mer ASASA-pIII phage display library 8 5e8 Markus F. Renschler Completely random Linear This library is based on fAFF1 vector, displaying N-terminal random octapeptides linked to pIII with the spacers ASASA. 194 8-mer P4-pIll and 8-mer P6-pIII phage display library 8 5e8 Markus F. Renschler Completely random Linear This library pool is based on fAFF1 vector, displaying N-terminal random octapeptides linked to pIII with the spacers polyproline (mixture of P4 and P6). 195 12-mer GG-pIII phage display library 12 5e8 Markus F. Renschler Completely random Linear This library is based on fAFF1 vector, displaying N-terminal random octapeptides linked to pIII with the spacers GG. 196 X9 phage display library 9 5e8 Algirdas J. Jesaitis Completely random Linear 197 TSAR-12 phage display library 20 Brian K. Kay Semi-random Linear The TSAR12 library contains a randomized peptide insert expressed at the N terminus of pIII, a minor coat protein of the M 13 filamentous phage. The displayed peptides are 23 aa long, and consist of an N-terminal sequence of ten randomized aa, a central sequence containing a fixed Gly residue flanked by two aa with partial variability, followed by ten additional randomized aa. 198 TSAR-9 phage display library (X18PGX18) 36 Brian K. Kay Semi-random Linear 199 [SC]X10[SC] phage display library 10 3.6e8 Peter S. Kim Completely random NNS Linear The library was constructed with 10-residue random insert flanked by Ser or Cys residues and can be expressed at the NH2-terminus of the pIll protein of the bacteriophage fd. 200 CX5C+CX6C+CX7C+CX9 phage display library pool 5, 6, 7, 9 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Completely random NNK 201 X2CX14CX2+X2CX18 phage display library pool 18, 20 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Semi-random NNK 202 X3YX4 phage display library 7 3e8 Hermann Gram Semi-random NNK Linear The X3YX4 phage display library is based on the phagemid vector pGEM-gIII, displaying randomized peptides fused to gpIII on the surface of M13 filamentous phage. The peptide library was randomized at seven positions surrounding an invariant tyrosine residue. 203 X5YSKPPPIP M13 phage display library 5 Leslie J. Berg Completely random Linear 204 X2CX4-8CX2 phage display library pool 10-14 3e8 De Ciechi Semi-random NNK Circular Five constrained libraries wherein random peptides, varying from four to eight amino acids, are flanked by a Cys on either side in addition to a further two random residues on either side were constructed with pMON20401 vector, which was derived from fUSE3 vector. The peptides are expressed immediately following the gene III signal peptide cleavage site. The codons of the degenerate oligonucleotides used for construction of the libraries followed the formula NNK, where N is an equimolar mixture of all four deoxynucleotides and K is an equimolar mixture of dGTP and dTTP. NH2-X2CX4-8CX2-GAAGGAGAGAG=TVES... 205 X10+X9GAX9 phage display library pool 10,18 2e8, 4e8 De Ciechi Semi-random Linear The 10-mer and 20-mer linear library were constructed with vector pMON6000 and pMON20401 respectively. Both vectors were derived from the fUSE3 vector. The peptides are expressed immediately following the gene III signal peptide cleavage site. The codons of the degenerate oligonucleotides used for construction of the libraries followed the formula NNK, where N is an equimolar mixture of all four deoxynucleotides and K is an equimolar mixture of dGTP and dTTP. NH2-X10-GG=TVES; NH2-X9GAX9-GAAGGAGAGAG=TVES...... 206 CX6C phage display library 6 C. Demangel Completely random Circular This library is constructed with the expression vector pC3H, which is derived from the phagemid pBluescript designed by Stratagen. A collection of oligonucleotides encoding random hexapeptides flanked by two cysteines and four constant residues EDGACXXXXXXCGAAS was inserted in the XhoI-SpeI cloning site. The insert was located between a leader sequence (pelB) and a truncated gene III, and allowed the monovalent display of random peptides in a constrained form on the N-terminus of the protein III. 207 X8 phage display library 8 Angus C. Nairn Completely random NNS Linear This library was constructed with vector M13CL2, which was derived from the phage vector M13mp18. Random peptide sequences were expressed on the N-terminus of the minor coat protein (gpIII). NH2-AX8GAAGAIEGR=AETV...GAAGA is the flexible-arm sequence and IEGR is the recognition sequence for Factor Xa. 208 X20 phage display library 20 1e8 Jonathan M. Gershoni Completely random NNK Linear This library was constructed in the fUSE5 vector. A 93 nucleotide long oligonucleotide encoding random 20 amino acid sequences was inserted into gene III at the region corresponding to the amino terminus of pIII. The oligonucleotides ON93 (5'-GAGCCAGTGCATCA(NNK)20TCGCTAACAGGTGGGTCTG-3') containing the degenerate sequences NNK20. 209 X10 fUSE5 phage display library 10 4.0e8 Philippe Valadon Completely random NNK Linear This decapeptide library was based on the fUSE5 vector. Random peptides were displayed at the N-terminal end of the pIII coat protein of the tetracycline-resistant phage fd. 210 CX7C and CX9C phage display library pool 7, 9 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Completely random NNK Circular The phage librarie were based on the fUSE5 vector. 211 X6 phage display library 6 Klaus Mosbach Completely random Linear pIII protein NH2-ADGAX6GTAG 212 D38 phage display library 35 5e8 Stephen J. McConnell Semi-random NNB This long peptide library was constructed in an M13 cloning vector (CYT-V1) that was generated by modifying the RF form of vector M663. The original M663 was derived from M13mp. The N-terminal pIII fusion proteins displayed in the D38 library are X20[YHND]A[IMTNKSR]X15. X residue is encoded by NNB. 213 DC43 phage display library 40 1e8 Stephen J. McConnell Semi-random This long peptide library was constructed in an M13 cloning vector (CYT-V1) that was generated by modifying the RF form of vector M663. The original M663 was derived from M13mp. The N-terminal pIII fusion proteins displayed in the DC43 library are X20GCGX20. X residue is encoded by NNB. 214 X10 phage display library 10 2e8 De Ciechi Completely random NNK Linear The 10-mer linear library was constructed with vector pMON6000 and presented on fd-tet. The peptides are expressed immediately following the gene III signal peptide cleavage site. NH2-X10-GG=TVES... 215 R8C phage display library 8 1.1e8 Heather Hanson Pierce Completely random Linear Two oligonucleotides were synthesized to generate the DNA cassette used in the construction of the conformationally constrained library. The first was a degenerate oligonucleotide, 5'-TGACGTCTCGAGTTGT(NNK)8-TGTGGATCTAGAAGGATC-3', containing an Xho I site, where N represents an equimolar mixture of A, C, G and T; and K corresponds to an equimolar mixture of G and T. The NNK coding scheme utilizes 32 codons to encode 20 amino acids; the frequency of each amino acid is once (Cys, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Trp, Tyr), twice (Ala, Gly, Pro, Val, Thr), or thrice (Leu, Arg, Ser) per codon. The second oligonucleotide, 3'-CCTAGATCTTCCTAG-5', contained an Xba I site, and served as a primer for DNA synthesis. 216 BPTI/Delta-g3p/biased 8-mer phage display library 4 6.2e7 Steven P. Weinheimer Completely random NNS Linear This substrate library was based on the phagemid vector pCANTAB5E. GGPGG-YLQAX4-GGPGG. The sequence YLQA is from the HSV-1 wild-type R cleavage site. YLQAX4 was flanked on either side by the amino acids GGPGG to disrupt any neighboring secondary structure that might influence protease cleavage. 217 BPTI/Dg3p/6-mer phage display library 6 6e8 Steven P. Weinheimer Completely random NNS Linear This substrate library was based on the phagemid vector pCANTAB5E. GGPGG-X6-GGPGG. The randomized insert was flanked on either side by the amino acids GGPGG to disrupt any neighboring secondary structure that might influence protease cleavage. 218 X10 phage display library 10 4e8 Christian (chiron) Completely random NNK Linear The random peptides were expressed on the protein PIII of bacteriophage fd (PMID: 1404385). 219 X8 phage display library 8 2e7 Joerg Koehl Completely random NNK Linear The library was based on the vector pCANTAB5 (Pharmacia). Random peptides were expressed on the protein PIII. 220 CWl phage display library 12 1e9 Naohiko Ikegaki Completely random NNK Linear The CW1 library is an X12 library with NNK coding scheme. The random peptides are displayed at the N-terminus of mature PIII. The library was constructed in a bacteriophage Ml3 vector, mBAX. 221 X21 phage display library 21 6.5e7 Anthony J. Conley Completely random NNK Linear The library was based on the vector fUSE5. Random peptides were expressed on the protein PIII. 222 CX5GPXRX5C bias phage display library 11 Anthony J. Conley Semi-random NNK Circular The library was based on the vector fUSE5. GPXR represents the V3 region apex sequence. Random peptides were expressed on the protein PIII. 223 LLX5GPXRX5LL bias phage display library 11 Anthony J. Conley Semi-random NNK Linear The library was based on the vector fUSE5. GPXR represents the V3 region apex sequence. Random peptides were expressed on the protein PIII. 224 CX5C phage libray 5 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Completely random Circular 225 X16 phage display library 16 Valery Alakhov Completely random NNK Linear The library containing 16-amino acid peptide was constructed , using fUSE5 as the phage vector. 226 X15 phage display library 15 2.0e7 Chiron (Emeryville, CA) Completely random Linear 227 C9C T7 phage display library 9 1.0e10 I. Todd Completely random Circular The peptides were expressed near the C-terminus of the T7 gene X surface coat protein (415 copies per phage). The random peptides of the T7 library were encoded by double stranded DNA inserts assembled from synthetic degenerate oligonucleotides and cloned into gene X of the vector (T7select415-1) (Bioscience, Cambridge, UK) at HindIII and EcoRI restriction sites. The vector DNA and insert DNA were ligated with T4 DNA ligase, and assembled into phage using T7Select packaging extract (BioScience, Cambridge, UK). The phage were amplified in E. coli BL21. 228 CX15C phage display library 15 1.8e8 Gardsvoll H Completely random Circular A random 15-mer constrained peptide library was constructed in the pComb8 phagemid, and consists of a random 15 amino acid sequence, flanked by two cysteine residues for a cyclic conformation. 229 CX7C+CX8C+CX10C+CX3CX3CX3C+CX3CX4CX2C phage display library pool 7-10 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Semi-random Circular 230 X9 λ phage display library 9 Gianni Cesareni (Tor Vergata University, Rome, Italy) Completely random NNK Linear The library of C-terminal random nonapeptides was constructed in lambda phage. 231 X6 phage display library 6 Pasqualini R, Arap W Completely random Phage display random peptide libraries based on vector fUSE5 displaying inserts with the general arrangementX6 (X-any amino acid residue) were designed and constructed with a diversity of 1.0e8 to 1.0e9 clones. 232 Tendamistat loop I phage display library (xQxxxxxxSx) 8 3.9e8 William F.DeGrado Semi-random NNS Linear The β-turn region between two anti-parallel β-strands on the loop I of tendamistat, an inhibitor of α-amylase, was extended by two residues and randomized in a phagemid library. 233 RGDX phage display library (xxRGDxxxxx) 7 3.2e8 William F.DeGrado Semi-random NNS Linear In library, the RGD motif was fixed at a particular position of loop I, with the libraries being designated as RGDX. Seven residues flanking the motif, including the conserved residues Q16 and S21, were randomized. 234 XRGD phage display library (xxxRGDxxxx) 7 1.4e8 William F.DeGrado Semi-random Linear In library, the RGD motif was fixed at a particular position of loop I, with the libraries being designated as and XRGD. Seven residues flanking the motif, including the conserved residues Q16 and S21, were randomized. 235 X6 phage display library 6 6.4e7 2.4e8 Jeffrey W. Smith Completely random NNK Linear Substrate phage display library pool were generated using a modified version of the fUSE5 phagemid. A FLAG epitope was engineered at the NH2 terminus of the geneIII protein by ligating them into fUSE5 at the KpnI and XbaI restriction sites. 236 X5 phage display library 5 3.2e6 1.8e8 David Deperthes Completely random NNS Linear Substrate phage display library pool were generated using a modified pH0508b phagemid. The construction consists of a His6 tag at either end of a Gly-Gly-Gly-Ser-repeat-rich region that precedes the carboxyl-terminal domain (codons 249-406) of the M13 gene III. 237 CX6C phage display library 6 1.0e6~1.0e7 Wadih Arap(The University of Texas M.D. Anderson Cancer Center, USA) Completely random Circular A phage display random peptide library based on the vector fUSE5 displaying the insert CX6C (C, cysteine; X, any amino-acid residue) was constructed with a size between 1 × 10e6 and 1 × 10e7. The fUSE5 plasmid is propagated in F'-minus host bacteria Escherichia coli MC1061. These bacteria should be grown in LB media (100 μg/mL streptomycin and 20 μg/mL tetracycline). The fUSE5 vector was engineered to contain a 14-bp “stuffer” that renders the phage noninfective by disrupting the gene III reading frame. 238 X12 phage display library 12 Lee KY Completely random Linear The phage display library was constructed from the pCANTAB5E phagemid vector (Pharmacia, Piscataway, NJ, USA) with 12 random amino acids, cyclized by flanking cysteines with the N-terminal extension AAQPACX12CAAA. The complexity of the library was approximately 1.0e8 with a titer of 1.0e11 cfu/mL. 239 pComb8 CX15C phage display library 15 Department of Biochemistry, University of Amsterdam, The Netherlands Completely random Circular 240 pIF4 X15 phage display library 15 P. Monaci (Instituto di Ricerche di Biologia Moleculaire [IRBM], Rome, Italy) Completely random Linear 241 pIF4 X28 phage display library 28 P. Monaci (Instituto di Ricerche di Biologia Moleculaire [IRBM], Rome, Italy) Completely random Linear 242 X10 phage display library 10 2e6 Fischer HD Completely random Linear The random decapeptides were produced as N-terminal fusions to the pIII surface protein of fd filamentous phage. The degenerate DNA inserts coding the decapeptides were chemically synthesized with an equal mixture of all four nucleotide bases at the three positions in each of the ten codons. 243 CPL4b phage display library (DGXXXCRGDCXXX) 13 1.0e6 Semi-random Circular Library CPL4b was based on vector fdVT3. The RGD motif library displaying the randomized peptides as g3p fusion protein is generated: CPL4b (DGXXXCRGDCXXX...) with the X residues (=L, V, F, Y,W, R, H, D, E, C, Q, S, G, A or P) encoded by the triplet BNK. 244 fUSE5-based X10 phage display library 10 Oddmund Bakke Completely random Linear 245 X4CX2GPX4CX4 phage display library 14 5e8 Brian C. Cunningham Semi-random Circular X4CX2GPX4CX4 library was designed to incorporate a type I beta-turn within the disulfide loop as observed in the bound conformation of a peptide agonist of the erythropoeitin receptor. Random peptides were displayed as N-terminal fusions linked through a glycine-rich spacer sequence to the multicopy Gene VIII phage coat protein. 246 X(i)CX(j)CX(k) phage display library pool 20 4e9 Brian C. Cunningham Semi-random NNS Circular Seven disulfide-constrained peptide libraries of the form X(i)CX(j)CX(k), with j values ranging from 4 to 10 and i+j+k=18 were generated and pooled. The seven libraries were X7CX4CX7, X7CX5CX6, X6CX6CX6, X6CX7CX5, X5CX8CX5, X5CX9CX4 and X4CX10CX4. Random peptides were displayed as N-terminal fusions linked through a glycine-rich spacer sequence to the multicopy Gene VIII phage coat protein. 247 CPL4c phage display library (XXXCRGDCXXX) 11 4.0e4 Semi-random Circular Library CPL4c was based on vector fdVT3. The RGD motif library displaying the randomized peptides as g3p fusion protein was generated: CPL4c (XXXCRGDCXXX...) with the X residues (=L, V, F, Y,W, R, H, D, E, C, Q, S, G, A or P) encoded by the triplet BNK. 248 X15 fUSE5 phage display library 15 2.5e8 Hideyuki Saya (Kumamoto University) Completely random NNK Linear The randomized peptide sequences in the library were displayed on a filamentous phage (fd phage) surface protein (pⅢ). 249 CX9C phage display library 9 1.6e6 M. A. A. Persson Completely random NNK Circular The fUSE 2 vector from filamentous phage vector fd-tet was modified as follows to obtain the vector fAST. 250 CX15C phage display library 15 1.8e6 M. A. A. Persson Completely random NNK Circular The fUSE 2 vector from filamentous phage vector fd-tet was modified as follows to obtain the vector fAST. 251 X15 phage display library 15 5.7e8 2.5e11 Ivone M. Takenaka Completely random NNM Linear The 15-mer phage display random peptide library was constructed using the fUSE-5 vector, kindly provided by G. Smith (University of Missouri). FUSE-5 replicative Form DNA was purified from K802 Escherichia coli and linearized by SfiI digestion, and the resulting vector DNA was ligated to synthetic DNA fragments containing complementary BglI termini. These duplex oligonucleotides contained (NNM)15 triplets in the coding strand, where N corresponds to equivalent amounts of A, T, G, and C and where M corresponds to equivalent amounts of G and T (minimizes occurrence of translational stop codons). The noncoding strand consisted of 18 or 45 residue stretches of the inosine nucleotide analog. Recombinant DNA was electroporated into MC1061 E. coli electrocompetent cells, spread on 94 plates (24 x 24 cm) with NZamine-yeast extract (NZY) agar plus 40 μg/ml tetracycline (tet) and incubated overnight at 37 °C. Phage were collected by polyethylene glycol precipitation and purified on cesium chloride gradients. The number of transducing units (TU) was determined by infecting K91Kan-resistant cells with an aliquot from each library and plating on tet plates. FUSE5-vector DNA containing an insert restores the reading frame of the pIII gene product, enabling the phage to infect K91Kan-resistant cells and produce colonies on tet plates. 252 XC(X)10CX phage display library 14 J. K. Scott Semi-random NNK Circular The library was constructed in the vector f88.4. 253 FMC12C phage display library 12 Completely random NNK Circular The library was constructed in a modified fd-tet vector. 254 X9 T7 phage display library 9 2e10 Lars Hellman Completely random NNK Linear The general structure of the aa sequence in the phage clone is PGG(X)9(H)6. 255 pComb M13 phage display library 6 5.0e6 6.4e7 Roger Y. Tsien Completely random Linear Amino acid sequences of the format Met-(His6)-(E)9-(X)6-(R)9 (where X 6 represents 6 randomized amino acids) were fused to the N terminus (0-4 copies per phage) of a truncated form of the M13 phage gIII coat protein. A hexahistidine motif was added at the N terminus to allow separation by immobilized metal affinity chromatography of unmodified phage from phage whose peptides had been cleaved releasing the hexahistidine tag. 256 CX10C T7 phage display library 10 Maria A. S. Pinhal Completely random NNK Circular 257 TN phage display library pool 12-18 Dyax Corp Semi-random Three disulfide-constrained cyclic peptide phage display libraries (TN6-6, TN10-9, and TN12-1) were pooled in approximately equal amounts. All of the cyclic peptide libraries (TN6-6, TN10-9, and TN12-1) were constructed in same manner, i.e. in a derivative of M13mp18, MANP, having the following modifications: bla (AmpR) gene, modified junction between signal of iii and coding region for mature III, and removal of LacZ complementation system. MANP comprises 8164 bases. The bla gene was from pGEM3Zf1(+), was bound by BamHI-HindIII sites at the 5' end and HindIII-SalI sites at the 3' end, replaced bases 6001 through 6432 of M13mp18, and was in the same orientation as the phage genes. The junction between the III signal sequence and mature III was modified with an NcoI restriction site embedded in the last three codons of the signal sequence. After the III signal, MANP contained the sequence of BPTI with restriction sites for the enzymes StyI, XhoI, PflMI, ApaI, Bsp120I, EcoO109I, PspOMI, BssHII, StuI, BstZ17I, BlpI, EagI, and SphI, which were all unique within MANP. Following BPTI, a unique PstI site and a Factor Xa cleavage site were inserted in a short linker region before mature III. The DNA encoding the library was synthesized with constant DNA on either side so that the DNA could be PCR amplified using TAQ DNA polymerase, cleaved with NcoI and PstI, and ligated to similarly cleaved vector. The variegated parts were synthesized with TRIM technology, which incorporates trinucleotides and allows mixtures of any set of amino acid types in any desired proportions. The flanking and varied DNA sequence positions of TN10-9 phage library is depicted as "AEGTGS-X1X1X2 CX3X3X3X3X3X3X3X3C X2X1X1-APGPTDS", where X1 belongs to any residue of DFHLNPRSWY, X2 denotes any residue of ADFGHLNPQRSVWY, and X3 is any natural amino acids except cysteine. More information on TN6-6 and TN12-1 phage library, check the library with ID 340 and 341 respectively. 258 Byungheon Lee (Department of Biochemistry and Cell Biology, School Medicine, Kyungpook National University, Republic of Korea) 7 5.0e8 In-San Kim, Byung-Heon Lee Completely random NNK Circular The phage display library is based on T7 415-1b phage vector. 259 T7 phage display library (X2PX4) 7 Shunsuke Kamijo Semi-random Linear The phage display library is based on T7 415-1b phage vector. 260 CX7C fUSE5 phage display library 7 Erkkl Ruoslahti Completely random NNK Circular 261 fUSE5-based X15 phage display library 15 2.5e8 Toshinori Sato Completely random NNK Linear 262 fUSE5-based X10 phage display library 10 1.4e9 R. Mandeville (Institute Armand-Frappier, University of Quebec, Laval, Canada) Completely random NNK Linear 263 X18 T7 phage display library 18 Thomas D. Wang Completely random NNK Linear The T7 library was constructed with the T7Select 10-3b vector as reported in the T7Select System Manual (Novagen, Gibbstown, NJ). 264 X12 M13 phage display library 12 Angela M. Belcher Completely random Linear 265 Phage display library ON159.3 (X12) 12 Stephen Albert Johnston Completely random NNK Linear The library was constructed in the vector fd-tet. 266 ON543 phage display library (X20) 20 Stephen Albert Johnston (Affymax, Palo Alto, CA) Completely random NNK Linear The library was constructed in the vector fd-tet. 267 X9 phage display library 9 R. Cortese (IRBM, Pomezia, Italy) Completely random Linear 268 JCFN-RGD phage display library (XRGDXXXX) 8 1.5e9 Semi-random NNK Linear The phage-display vector for FNfn10, JCFN, was constructed by cloning the FNfn10 gene7 in the modified M13 vector, JC-M13-88. A FNfn10 library, JCFN-RGD, was constructed in such a way that the FG loop has XRGDXXXX sequence where X stands for any amino acid (residues 77–84). Mutagenesis on the JCFN template was performed using an oligonucleotide JCFNFGRGD (GTTAATCGAGATTGGCTTGGAMNNMNNMNNMNNATCGCCGCGMNNAGTAACAGCGTATAC, where N is a mixture of A, G, C and T, and M is a mixture of A and C). 269 X15 T7 phage display library 15 1.7e10 Susumu Kobayashi, Fumio Sugawara, Kengo Sakaguchi Completely random NNK Linear The T7 library was constructed with the T7Select 10-3b vector. 270 X12 phage display library 12 Yasser Perera Completely random Linear 271 XCX(3)SDLX(3)CI phage display library 13 J. K. Scott Semi-random NNK Circular 272 X(7)SDLX(3)CI phage display library 15 J. K. Scott Semi-random NNK Circular 273 X12 T7 phage display library 12 7.0e8 Toshiyuki Mori Completely random NNK Linear The T7 library was constructed with the T7Select 10-3b vector. 274 CX5C T7 phage display library 5 1.2e9 Toshiyuki Mori Completely random NNK Circular The T7 library was constructed with the T7Select 10-3b vector. 275 CX7C T7 phage display library 7 1.4e9 Toshiyuki Mori Completely random NNK Circular The T7 library was constructed with the T7Select 10-3b vector. 276 CX6C T7 phage display library 6 1.8e9 Toshiyuki Mori Completely random NNK Circular The T7 library was constructed with the T7Select 10-3b vector. 277 fNG1 phage display library (X30) 30 Y.-C. JACK CHEN Completely random NNK Linear The vector fNG1 was derived from M13tsp3. The vector M13tsp3 is a derivative of M13mpl8 having inactivated the a-complementing fragment of lacZ of M13mpl8 and to which the Ap(R)-conferring gene was added. The Ap(R) marker aids in identification of transformants and applies a selective pressure for maintaining the vector in the cells. 278 X8 fAFF1 phage display library 8 1.4e9 W. Dower (Affymax) Completely random NNK Linear The library is based on a filamentous bacteriophage affinity vector, fAFF1, which was constructed from the tetracycline resistance-transducing vector fd-tet. Random peptides were express on protein III. Coding scheme: NNK, where N represents equimolar A, C, G, or T and K is equimolar G or T. NH2-XXXXXXXXGG=TVESC, amino acids after the equals sign belong to the native gene III protein. 279 SGTACX(2)GPX(4)CSLAGSP M13 phage display library 20 1.8e8 Henry B. Lowman Semi-random NNS Circular 280 X(4)CX(2)GPX(4)CX(4) M13 phage display library 18 7.9e8 Henry B. Lowman Semi-random NNS Circular 281 X20 M13 phage display library 20 5.0e8 Henry B. Lowman Completely random NNS Linear 282 T7 CX7C phage display library 7 Erkki Ruoslahti Completely random NNK Circular 283 CX(2)GPX(4)C, X(4)CX(2)GPX(4)CX(4), and X(i)CX(j)CX(k) M13 phage display library pool 8, 18, 20 Mark S. Dennis Semi-random NNK Linear For X(i)CX(j)CX(k) phage display library, j=8-10, i+j+k=18 and |i-k|<2. 284 X20 and X(i)CX(j)CX(k) M13 phage display library pool 20 Mark S. Dennis Semi-random NNK Linear For X(i)CX(j)CX(k) phage display library, j=4-7, i+j+k=18 and |i-k|<2. 285 X8 and X(2)CX(j)CX(2) M13 phage display library pool 8, 10-12 Mark S. Dennis Semi-random NNK Linear For X(2)CX(j)CX(2) phage display library, j=4-6. 286 X(2)CX(j)CX(2) M13 phage display library 13-16 Mark S. Dennis Semi-random NNK Circular For X(2)CX(j)CX(2) phage display library, j=7-10. 287 X(i)CX(j)CX(k) M13 phage display library pool 20 4.0e9 James A. Wells Semi-random NNS Circular For X(i)CX(j)CX(k) phage display library pool, i+j+k=18. 288 f8 α phage display library (XXLXXXAX) 8 V. A. Petrenko and G. P. Smith Semi-random NNK Linear The alpha library is a new type of phage display library, in which biological selection helps to generate a great variety of conformationally biased α-helical ligands. The library was constructed in the vector f8–5 (GenBank Accessions bankit439334) 289 X15 PC89 phage diaplay library 15 Stefan Wagner Completely random NNN Linear 290 CX7C, CX8C, and CX9C FUSE5-based phage display library pool 7-9 Mikhail G. Kolonin Completely random NNK Circular Random peptide libraries were constructed in the bacteriophage vector fUSE5. 291 T7 phage display library Kei-ichi Takata Completely random Linear 292 X15 PC89 phage display library 15 Tamara Men????ndez (Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana, Cuba) Completely random Linear 293 X7 T7 phage display library 7 5.0e8 Erkki Ruoslahti Completely random Linear 294 IR20 phage display library (X20) 20 4.0e7 Kathlynn C. Brown Completely random NNS Linear A new library of random 20-mer peptides in a phage vector was created by cloning the sequence into bacteriophage vector fAFF-1. A library of 4.0e7 independent clones encoding 20-mer peptide sequences embedded between the BspEI and Acc65I cloning sites of fAFF-BA was constructed. This 20-mer library, referred to as IR20, differs from the original library in that the cloning region within the bacteriophage vector has been modified. This allows for efficient directional cloning and results in the addition of Ser-Gly to the N-terminus of the peptide-pIII fusion. In the previously used library, the 20-mer library is located directly at the N-terminus of the fusion, and it is possible that modification of the N-terminal amino group of the peptides may disrupt cell targeting. 295 X12 phage display library 12 Han-Chung Wu Completely random Linear 296 XXCX4CXX phage display library 10 3.0e8 Chi-Bom Chae Semi-random NNK Circular Random nucleotides were introduced into the NH2-terminal region of the pⅢ gene of the pCANTAB5E phagemid using SfiI and NotI sites. Each phage displays an octapeptide epitope containing a disulfide bond as fused to the NH2 terminus of pⅢ coat protein in M13 phage and contains a factor Xa cleavage site between an epitope and pⅢ coat protein. 297 pGWX3YX4 phage display library 8 1.1e8 Su-Jun Deng, Gaochao Tian Semi-random NNK Linear The library was constructed in the vector pGWg3. To construct phage display vector pGWg3, EcoRI- and HindIII-digested pUC18 was ligated with the M13 phage gIII fragment after being amplified using PCR with oligonucleotides 5’-GGGGAATTCA AACTCAGATC TGGTGGCGGT GGATCCCCAT CGTTTGTGAA TATCAAGGC-3’ and 5’-GCCAAGCTTC TAATAATAAC GGAATACCCA AAAGAACTG-3’. Subsequently, the small PvuII fragment of the ligated product that contained gIII was inserted into the large fragment of PvuII-digested pBluescript. A DNA fragment encoding Pel B leader peptide and multiple cloning sites was constructed by a synthetic ligase chain reaction approach using 4 oligonucleotides 5’-AATTGAATAG AGGGTAGAAT TGAAATACCT GCCGACCGCT GCTGCTGGTC TGCTCCTCGC GGCCC-3’, 5’-AGCCGGCCAT GGCCCAACTC GAGTGGGCGG CCGCAGGTGG ATAACAGAAA CTGATCTCCG AAGGGTACCA AGACCTGAAC A-3’, 5’-GATCTGTTCA GGTCTTGGTA CCCTTCGGAG ATCGTTTCT GTTATCCACC TGCGGCCGCC CACTCGAGTT GGGCCA-3’, and 5’-TGCCGGCTGG GCCGCGAGGA GCAGACCAGC AGCAGCGGTC GGCAGCAGGT ATTTCATCAA TTCTACCCTC TATTC- 3’. Insertion of this DNA fragment into the EcoRI- and BglII-digested pBluescript containing gIII yielded the phage display vector, pGWg3. 298 pGWX4YX4 phage display library 9 1.75e8 Su-Jun Deng, Gaochao Tian Semi-random NNK Linear The library was constructed in the vector pGWg3. To construct phage display vector pGWg3, EcoRI- and HindIII-digested pUC18 was ligated with the M13 phage gIII fragment after being amplified using PCR with oligonucleotides 5’-GGGGAATTCA AACTCAGATC TGGTGGCGGT GGATCCCCAT CGTTTGTGAA TATCAAGGC-3’ and 5’-GCCAAGCTTC TAATAATAAC GGAATACCCA AAAGAACTG-3’. Subsequently, the small PvuII fragment of the ligated product that contained gIII was inserted into the large fragment of PvuII-digested pBluescript. A DNA fragment encoding Pel B leader peptide and multiple cloning sites was constructed by a synthetic ligase chain reaction approach using 4 oligonucleotides 5’-AATTGAATAG AGGGTAGAAT TGAAATACCT GCCGACCGCT GCTGCTGGTC TGCTCCTCGC GGCCC-3’, 5’-AGCCGGCCAT GGCCCAACTC GAGTGGGCGG CCGCAGGTGG ATAACAGAAA CTGATCTCCG AAGGGTACCA AGACCTGAAC A-3’, 5’-GATCTGTTCA GGTCTTGGTA CCCTTCGGAG ATCGTTTCT GTTATCCACC TGCGGCCGCC CACTCGAGTT GGGCCA-3’, and 5’-TGCCGGCTGG GCCGCGAGGA GCAGACCAGC AGCAGCGGTC GGCAGCAGGT ATTTCATCAA TTCTACCCTC TATTC- 3’. Insertion of this DNA fragment into the EcoRI- and BglII-digested pBluescript containing gIII yielded the phage display vector, pGWg3. 299 T7 phage display library Dennis E Hallahan Completely random Linear 300 X15CX phage display library 17 J. K. Scott (Simon Fraser University, Burnaby, Canada) Semi-random NNK Linear The library was based on PC89 vector. 301 X16 mRNA display library 16 Nobuhide Doi Completely random NNS Linear The 16-mer random DNA library was amplified from G4SG4S(NNS)16FLAGA6r by PCR using priSP6OGf and priFLAGA6r primers. 302 X(2)-F-X(3)-W-X(2)-L-X(2) mRNA display library 12 Nobuhide Doi Semi-random NNS Linear The 12-mer random DNA library was amplified from X12(FWL)-r using 5' o29-T7-EcoRI and Flag1A-lib primers. 303 X(3)-L-I-X-E-I-X(2)-I-L-X(2)-I-X(3)-L mRNA display library 19 Teruaki Kobayashi Semi-random NNK Linear CA11, a IL-6-binding peptide, was isolated from a random-primed human cDNA library. The X(3)-L-I-X-E-I-X(2)-I-L-X(2)-I-X(3)-L mRNA display library was constructed based on CA11. 304 X16 mRNA display library 16 6.6e20 Hiroshi Yanagawa Completely random NNS Linear Random lib-Fw (a plus chain encoding SP6 promoter, Ω29 sequence, start codon, and a part of G/S linker), and Random lib-Rv (a minus chain encoding a G/S linker, randomized region, FLAG-tag, and A 6 sequence) were used for a wholly random library (library A) containing random 16-aa sequences encoded by (NNS)16 codons. 305 X(2)-V-X-R-X-L-X(4)-D-X-I-X(2) mRNA display library 16 2.0e14 Hiroshi Yanagawa Semi-random NNS Linear Bak lib-Fw (a plus chain encoding T7 promoter, SD sequence, start codon, and a part of G/S linker) and Bak lib-Rv (a minus chain encoding a G/S linker, Bak library, FLAG-tag, and A 6 sequence) were for a partially randomized library (library B), in which 5 residues of the Bak-BH3 sequence were conserved within the 16 residues. 306 X10 mRNA display library 10 Tatsuro Shibui Completely random Linear The template DNA for the library was divided into three pieces and synthesized chemically. These fragments were then connected by annealing, the gaps filled and the DNA amplified with PCR. The constructed DNA templates contain a SP6 promoter and IRES and the coding regions for a random 10-amino acid peptide and FLAG-tag. In order to minimize the appearance of stop codons in the random sequence, a four-nucleotide ratio in each of the first, second, and third letters in codons (PQK)10 was set up (P = T:C:A:G = 15:25:30:30, Q = T:C:A:G = 30:30:30:10, and K = T:C:A:G = 50:0:0:50). 307 [RKEDG]-C-X(10)-C mRNA display library 13 Jack W. Szostak Semi-random NNB Circular 30-nt region (NNB)10 , encoding a random 10-mer peptide, flanked by cysteine codons TGC. The choice of NNB random triplets decreases the number of stop codons in the random portion. The 3' constant region encodes a GSVG spacer and 6 histidines (his(6)-tag). The nine nucleotides downstream of the his 6 -tag are complementary to the 2'-O-Me RNA portion of the cross-linking puromycin-terminated oligonucleotide. They encode the tripeptide HRL, which is followed by a TAG stop codon. 308 YAC-TGZ-(XYZ)18-YAC mRNA display library 21 LIN XiuKun Semi-random Linear The synthesized DNA comprised 21 consecutive random codons, and these random codons were flanked by constant sequences. The 5' primer contained a T7 RNA polymerase promoter, a tobacco mosaic virus (TMV) translation enhancer sequence, and a start codon. The 3' primer included sequences coding for His(6) tag and a complementary sequence for linking with puromycin linker. 309 X20 mRNA display library 20 5.2e12 Rihe Liu Completely random Linear Each sequence in the DNA library contains a T7 RNA polymerase promoter, a TMV translation enhancer sequence, an N-terminal FLAG tag coding sequence, a random cassette encoding 20 consecutive random codons, and a His(6) tag coding sequence. The codon mixtures referred to this had the following nucleotide distributions, as determined by sequencing individual clones. Position 1: 35% A 20% T 27% G 18% C; Position 2: 33% A 29% T 21% G 17% C; Position 3: 22% T 49% G 29% C. 310 M-X(4)-CRATKML mRNA display library 12 1.6e5 David C.H. Yang Semi-random NNS Linear The DNA template contained a T7 promoter, a deletion mutant of 5\'-untranslated region of tobacco mosaic virus 5\'-UTR (ΔTMV) for in vitro transcription and translation, respectively. The reading frame in the DNA template contained four random amino acid residues appended to the N-terminus of a constant sequence. The constant region encoded the amino acid sequence CRATKML, which was derived from the C-terminus of the SNAP-25, and is a known inhibitor of BoNT/A. 311 X27 mRNA display library 27 7.0e13 Richard W. Roberts Completely random Linear The anti-sense DNA oligo 130.2 [5'-AGC GCA AGA GTT ACG CAG CTG (SNN)27 CAT TGT AAT TGT AAA TAG TAA TTG TCC C; S = C or G, N = A, C, G or T] was PCR amplified with primers 47T7FP (5'-GGA TTC TAA TAC GAC TCA CTA TAG GGA CAA TTA CTA TTT ACA ATT AC) and mycRP (5'-AGC GCA AGA GTT ACG CAG CTG) to produce the initial template containing a T7 promoter, a 5'-untranslated region (UTR), an ATG methionine start codon, 27 random amino acids and a constant 30-end that encoded the peptide, QLRNSCA. 312 GPR X(6) mRNA display library 6 Richard W. Roberts Completely random Linear The C-GPR extension library was generated by PCR amplification of the template C-GPR-X6 (5a???2-AGC GCA AGA GTT ACG CAG CTG (SNN)6 CCG TTG ATC GTC CAG CCG TTT GGA CTG AGA CAT TGT AAT TGT AAA TAG TAA TTG TCC C; S = C or G) with primers 47T7FP and mycRP. The purified (QIAquick PCR purification) dsDNA constructs contained a T7 promoter, an untranslated region, and an ORF containing a 3a???2 constant sequence encoding the peptide QLRNSCA. 313 X(5)-C-X(5) mRNA display library 11 3.0e12 Richard W. Roberts Semi-random NNS Linear A synthetic DNA library template sd7 (ACTATTTACAACCACCATG-(NNS)5-TGC-(NNS)5-GGCGGCGACTAAGGACGACGATGACAAGGCGGCGGCGGC) was purified by preparative polyacrylamide gel electrophoresis and amplified by polymerase chain reaction (PCR) with the primers sd2 (GGATTCTAATACGACTCACTATAGGGACAATTACTATTTACAACCACCATG) and sd3 (GCCGCCGCCGCCCTTGTCATCGTCGTCCTTGTAGTC). 314 X15 mRNA display library 15 1.2e11 Michael Famulok Completely random Linear The random region was designed for low stop codon frequency and increased cysteine probability (1 Cys within the random region). The phosphoramidites of A, G, C, and T were dissolved in acetonitrile and then mixed for each codon position as follows: 5, A 19.2%, G 23.4%, C 23.4%, T 34.0%; 6, A 33.2%, G 32.9%, C 13.5%, T 20.4%; and 7, A 0.00%, G 31.0%, C 34.5%, T 34.5%. 315 X10 mRNA display library 10 9.0e12 6.9e13 Richard W. Roberts Completely random NNS Linear Library was constructed by inserting NNS codons at residues 13-22 in the N-myc template. 316 EETI-II mRNA display library (X6) 6 6.4e7 6.0e11 Richard W. Wagner Completely random NNS Linear The double helix mRNA/DNA strand encodes for the tobacco mosaic virus untranslated region and the 28 amino acids of the EETI-II library. The single-stranded poly-A linker is a covalent ~ 100 Å extension of the mRNA strand. The three prime end of the poly-A linker terminates with puromycin, which forms the carboxy-terminus of the peptide encoded by the mRNA. 317 c-myc mRNA display library (X27) 27 2.0e13 Richard W. Wagner Completely random NNS Linear The double helix mRNA/DNA strand encodes for the tobacco mosaic virus untranslated region and the 27 amino acids of the randomized linear peptide library. The single-stranded poly-A linker is a covalent ~ 100 Å extension of the mRNA strand. The three prime end of the poly-A linker terminates with puromycin, which forms the carboxy-terminus of the peptide encoded by the mRNA. 318 [VGAED]-X(14) ribosome display library 15 2.0e13 Volker A. Erdmann Completely random NNS Linear A DNA library encoding FABP, possessing 15 randomized amino acid residues at its N terminus instead of the four natural residues, was generated by introducing a DNA fragment encoding a GNN codon followed by 14 NNS codons into the FABP gene through DNA ligation. The construct used for ribosome display, contains all necessary features; the T7 promoter on the DNA level, and the 5' and 3' stem-loops stabilizing the mRNA against ribonucleases on the RNA level as well as the Shine-Dalgarno sequence for efficient in vitro translation. The 5'-untranslated region of the mRNA is derived from gene 10 of phage T7 and is capable of forming a stable stem-loop structure. The 3' stem-loop comes from a modified lipoprotein terminator (lpp) from Escherichia coli. 319 X12 ribosome display library 12 Xiao-Lian Zhang Completely random NNM Linear A tac promoter sequence and a ribosome-binding site (RBS) were followed by the sequence encoding the protein to be displayed, to effect strong transcription. In addition, at both ends of the mRNA, 5'-stem-loops and 3'-stem-loops were included in the ribosome display system to stabilize mRNA against RNase digestion. 320 X20-[YHND] ribosome display library 21 Sachiko Machida Semi-random NNB Linear This random peptide library was inserted into Cassette 1 and ligated to the other cassettes, followed by prescreening using the FLAG tag to exclude any sequences that did not express the full-length peptide; following prescreening, the library diversity was 1.0e8 per μg DNA. 321 ZXXXZXXZXXXZXX ribosome display library 14 Sachiko Machida Semi-random NNB Linear cDNAs encoding ZXXXZXXZXXXZXX (where Z represents K/R, and X represents any amino acid) were inserted into Cassette 1. 322 X(2)-C-X(10)-C-X(2) ribosome display library 16 Shinya Y. Sawata Semi-random NNY Circular The construct carried a T7 promoter (T7) and a Shine-Dalgarno sequence (SD) necessary for in vitro transcription and translation, respectively. The APL consisted of 14 NNY codons and a pair of TGT codons and was inserted between SfiI restriction sites, followed by the encoding sequences for C-variant RNA (Cv)-associating protein (Cvap) and a protein spacer (Ps). The stop codons were removed from the entire sequence for stalling a ribosome on mRNAs. 323 X10 ribosome display library 10 Yoshihiro Shimizu Completely random NNK Linear A gene for the spacer sequence [protein D (pD), a phage Lambda capsid protein] and the subsequent SecM arrest sequence was sub-cloned into pURE1 vector (post-genome institute) between BamHI and XhoI site. The resultant plasmid was used for the amplification of the library that encodes 10 amino acid random peptide sequences followed by the spacer sequence. Using 10_aa_library primer (5'-GAAGGAGATATACCATATG-(NNK)10-GGATCCGGTGGCGGTTCCG-3'; N is any nucleotide and K is either G or T, respectively) and SecM_25_RD_RT3 primer (5'-AAGCTTCTCGAGCCCGGTGAGGCGT-3'), a library sequence was amplified and purified by gel electrophoresis. The products were used as templates for the second step PCR. Using Universal primer (5'-GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCA-3') and SecM_25_RD_RT3 primer, a library was amplified and purified by gel electrophoresis. The products were used as templates for the ribosome display selection in the PURE system. 324 X9 E. coli bacterial display library 9 4.0e7 4.0e9 Per Klemm Completely random NNB Linear Random peptide library based on oligonucleotides 33 bp in length with BglII overhangs was constructed for display in the type 1 fimbria adhesin FimH. Vector (pLPA30) contained the fimH gene with a BglII linker inserted at codon position 225 and under the transcriptional control of the lac promoter. The inserted double-stranded oligonucleotides consisted of nine random codons flanked by BglII restriction sites (encoding Arg-Ser). Due to the presence of BglII overhangs, various numbers of double-stranded oligonucleotides were inserted in fimH, further adding to the complexity of the library. To express FimH variants as constituents of fimbriae, an auxiliary plasmid (pKKL115), containing all fim genes except fimH, was used for transcomplementation of the fimH-containing plasmid. Expression from the binary plasmid system led to display of chimeric FimH in the context of fully functional fimbriae. 325 X15 E. coli bacterial display library 15 5.0e10 Patrick S. Daugherty Completely random NNS Linear E.coli OmpA was chosen as a display scaffold. To maximize library construction efficiency, asymmetric SfiI restriction sites were introduced into an OmpA expression vector immediately preceding loop 1 and following loop 4. DNA fragments containing the random epitope insertions were synthesized by polymerase chain reaction (PCR), digested with SfiI, ligated into the display vector and transformed into the E.coli strain MC1061, which can be made highly transformation competent and is ara(-), allowing the use of the araBAD promoter for controlled OmpA expression. 326 FliTrx bacterial display library (X12) 12 1.77e8 2.0e10 Invitrogen, Carlsbad, CA Completely random Linear The FliTrx(TM) Random Peptide Display Library is an E. coli-based system that eliminates the use of phage and makes screening of peptide interactions faster and easier. The FliTrx(TM) Library was constructed in the pFliTrx(TM) vector. A diverse library of random dodecapeptides is positioned in the active site loop of the thioredoxin protein (trxA), inside the dispensable region of the bacterial flagellin gene (fliC). The resultant recombinant fusion protein (FLITRX) is exported and assembled into partially functional flagella on the bacterial cell surface. The FliTrx library, which expresses random dodecapeptides flanked by cysteine-glycine-proline/glycine-proline-cysteine sequences (CGP/GPC) on the cell surface as FliC-Trx fusions. 327 OmpX7C bacterial display library ( X(2)-C-X(7)-C-X(2) ) 13 4.0e9 Patrick S. Daugherty Semi-random NNS Circular Peptide insertion library within OmpX of the form X(2)-C-X(7)-C-X(2) was constructed using PCR with primers PD671/PD516, using as a template pB33OmpXtemp. The resulting products were digested with SfiI and ligated into SfiI-digested pB33OmpXT2. Ligation products were desalted and electroporated into electrocompetent MC1061 yielding 4.0e9, For CPX library construction, a PCR template was created (pB33CPX-template) lacking a passenger peptide and incorporating a silent mutation that destroys the SfiI restriction site. 328 OmpX3C bacterial display library ( X(4)-C-X(3)-C-X(4) ) 13 1.3e9 Patrick S. Daugherty Semi-random NNS Circular Peptide insertion library within OmpX of the form X(4)-C-X(3)-C-X(4) was constructed using PCR with primers PD671/PD516, using as a template pB33OmpXtemp. The resulting products were digested with SfiI and ligated into SfiI-digested pB33OmpXT2. Ligation products were desalted and electroporated into electrocompetent MC1061 yielding 1.3e9, For CPX library construction, a PCR template was created (pB33CPX-template) lacking a passenger peptide and incorporating a silent mutation that destroys the SfiI restriction site. 329 CX7(1)C bacterial display library ( X(2)-C-X(7)-C-X(2) ) 13 4.0e8 Patrick S. Daugherty Semi-random NNS Circular CX7(1)C bacterial display library was generated in a CPX vectors. PCR was performed with primers PD707 and PD634 using pB33CPX-template. The resulting PCR product was lengthened in a second PCR to enable efficient digestion, using primers PD180 and PD753. This product was digested with SfiI and ligated into HincII/SfiI digested pB33CPX-template. 330 CX7(2)C bacterial display library ( X(2)-C-X(7)-C-X(2) ) 13 1.0e10 Patrick S. Daugherty Semi-random NNS Circular CX7(2)C bacterial display library was constructed using a CPX scaffold containing the substitutions L(-17)V, L(-14)V, L(-10)V, L26V, L37I, L113V, L123V, where the residue numbering is based on the wild-type OmpX. A plasmid encoding a CPX without leucine codons (pB33NLCPX) was constructed by overlap PCR. PCR products generated with primers PD515/PD703, PD704/PD632, and PD633/PD634 were used for overlap PCR with outside primers PD515/PD634. The resulting product was cloned into KpnI/HindIII digested pBAD33. The CX7(2)C bacterial display library was then constructed by PCR amplification of pB33NLCPX with primers PD707/PD180, amplification of the product with PD753/PD180, digestion with SfiI, and ligation into SfiI-digested pB33NLCPX. 331 X15 CPX bacterial display library 15 Patrick S. Daugherty Completely random NNS Linear X15 CPX bacterial display library was constructed as fusions to the extracellular N-terminus of a circularly permuted variant of outer membrane protein OmpX (CPX). Following the native signaling sequence of OmpX, CPX was constructed by fusion of the native C- and N-terminus of OmpX with a GGSG linker, and by opening loop 2 between residues S53 and S54 yielding a scaffold with extracellular C- and N-termini. 332 CX9C E. coli bacterial display library 9 Sang J. Chung Completely random NNN Linear The FcBP1-eGFP vector, which encodes a fusion protein of eGFP with FcBP1, at the C-terminus of FcBP1, was newly designed and constructed by PCR. An FcBP-eGFP DNA library was constructed by inserting 27 nt-randomized DNA sequences into the N-terminus of the eGFP gene by PCR using the eGFP gene as template under the conditions described above (50-primer: 50-GCC GCA GCC ATA TGA AAG ATG AAT GTN NNN NNN NNN NNN NNN NNN NNN NNN NNT GCG AAT TCG AGC TCC GTC GA-30; 30-primer, ATT CGC GGA TCC CTT GTA CAG CTC GTC CAT GCC GAG; where N is A, T, G, or C, and the restriction sites for NdeI and BamHI are italicized). The purified PCR product was digested with NdeI and BamHI and cloned into pHCE-IIB, yielding the FcBP-eGFP DNA library. 333 PL12 yeast display library (X12) 12 Angela M. Belcher Completely random NNK Linear A yeast display peptide library was created from a degenerate oligonucleotide pool encoding random 12-mer peptides with a NNK bias. Peptides were displayed as fusions to the C-terminus of Aga2, which is encoded on a plasmid downstream of a Gal-based promoter. The expression vectors were maintained in S. cerevisiae strain EBY100, which has Aga1 under control of a Gal-based promoter integrated in its genome, Oligonucleotides used in library construction were obtained from Oligos, Etc. The degenerate oligonucleotide pool encoding the random 12-mer peptide library (PL12LibrBstXI) 50-pGTGGCGGTAGCGGC-(NNK)12-TAGCTAGCTAGGCCAGTAGC, synthesized with an NNK bias in order to reduce the stop codon frequency, was annealed to two short primers (5'BstXIanneal) 5'-pGCCGCTACCGCCACCGCC and (3'BstXIanneal) 5'-pCTGGCCTAGCTAGCTA. The resulting hybrid DNA library containing duplexed BstXI sticky ends was directly ligated into the BstXI sites of pBPZ, and electroporated into E. coli Electromax DH10B (Invitrogen, Carlsbad, CA) for amplification on plated LB Ampt media. 334 LAP yeast display library 12 Alice Y. Ting Semi-random Linear The LAP library is displayed on the yeast surface as a fusion to Aga2p protein. A C-terminal myc epitope is used to quantify LAP expression level. A partially randomized oligo with the following sequence: 5'A AAT AAG CTT TTG TTC GGA TCC NGM MNN NAN NTS MNN MNN AAC TTT ATC MNN NTS NAN TCC GCT AGC CGA CCC TCC, was ordered from IDT (Integrated DNA technologies). N designates an equimolar mixture of all bases. S designates a 1:1 mixture of G and C. M designates a 1:1 mixture of A and C. This oligo was annealed with another oligo, Con2For.F (5'CT AGT GGT GGA GGA GGC TCT GGT GGA GGC GGT AGC GGA GGC GGA GGG TCG GCT AGC GGA), which overlaps with both pCTCON2 vector and the library oligo. The 5' overhangs were filled in using Klenow polymerase. The resulting product was PCR-amplified using the primers Con2For.F and Con2Rev.R (5'TA TCA GAT CTC GAG CTA TTA CAAGTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC). Meanwhile, pCTCON2 vector was prepared by digestion with NheI and BamHI, and gel-purified. PCR insert and pCTCON2 vector were transformed together into S. cerevisiae EBY100 (Invitrogen) by electroporation as described by Colby et al.52 Homologous recombination occurred inside the yeast. Serial dilutions of transformed yeast were plated on SDCAA plates and colonies were counted, to determine transformation efficiency. 335 SC-X(16)-C M13 phage display library 16 Mark W. Grinstaff Completely random NNK Circular 336 X8 M13 phage display library 8 Angela M. Belcher Completely random NNM Linear The M13KE phage vector was modified by making a cloning site for pVIII display. A Pst I restriction site was made by mutating T to A at position 1372, a BamH I site was made by mutating C to G at positon 1381, and the Pst I site at position 6246 was deleted by mutating T to A at position 6250. The site-directed mutagenesis was done using overlap extension PCR. A dsDNA library was then prepared and cloned into the resulting modified phage vector, named M13SK, using Pst I and BamH I. To obtain the dsDNA library, partial library duplexes were formed by annealing of extension primer (5\'-GATGCTGTCTTTCGCTGCAG-3\') with oligonucleotides (3\'-ACGACAGAAAGCGACGTCnm(nnm)6nnCCTAGGAACATC ATC-5\', where n=A, T, C, or G and m=A or C). The partial library duplexes were incubated with Klenow fragment (3\'→'5\' exo-) (10 U/μL) and dNTP at 37 °C for 30 min. The Klenow fragment was inactivated by heating (75 °C for 20 min), and the mixture was digested with Pst I and BamH I. The digested DNA was gel purified (2-40% TBE polyacrylamide gel), ligated into M13SK, and transfected to XL1-Blue Electroporation Competent Cells using a MicroPulser (Biorad). The library was titered according to manufacturer directions and sequenced (MIT Biopolymers Laboratory) before amplification. 337 X9 fUSE5 phage display library 9 Po Tien Completely random NNK Linear The plasmid was constructed in such a way that only clones bearing frame-restoring inserts could form infectious particles in this library. A large number of random 27-mers were synthesized and digested by BglI before they were inserted near the amino terminus of the minor coat-protein gene (gIII) of fUSE5. A phage particle could only express one of the random 27-mer sequences on its surface as a fusion protein of pIII. 338 X6 fUSE5 phage dislpay library 6 8.6e8 2.5e13 Ivone M. Takenaka Completely random NNM Linear The 6-mer phage display random peptide library was constructed using the fUSE-5 vector, kindly provided by G. Smith (University of Missouri). FUSE-5 replicative Form DNA was purified from K802 Escherichia coli and linearized by SfiI digestion, and the resulting vector DNA was ligated to synthetic DNA fragments containing complementary BglI termini. These duplex oligonucleotides contained (NNM)6 triplets in the coding strand, where N corresponds to equivalent amounts of A, T, G, and C and where M corresponds to equivalent amounts of G and T (minimizes occurrence of translational stop codons). The noncoding strand consisted of 18 or 45 residue stretches of the inosine nucleotide analog. Recombinant DNA was electroporated into MC1061 E. coli electrocompetent cells, spread on 94 plates (24 x 24 cm) with NZamine-yeast extract (NZY) agar plus 40 μg/ml tetracycline (tet) and incubated overnight at 37 °C. Phage were collected by polyethylene glycol precipitation and purified on cesium chloride gradients. The number of transducing units (TU) was determined by infecting K91Kan-resistant cells with an aliquot from each library and plating on tet plates. FUSE5-vector DNA containing an insert restores the reading frame of the pIII gene product, enabling the phage to infect K91Kan-resistant cells and produce colonies on tet plates. 339 TN6-6 phage display library 12 Dyax Corp Semi-random The disulfide-constrained cyclic peptide phage display library TN6-6 was constructed in a derivative of M13mp18, MANP, having the following modifications: bla (AmpR) gene, modified junction between signal of iii and coding region for mature III, and removal of LacZ complementation system. MANP comprises 8164 bases. The bla gene was from pGEM3Zf1(+), was bound by BamHI-HindIII sites at the 5' end and HindIII-SalI sites at the 3' end, replaced bases 6001 through 6432 of M13mp18, and was in the same orientation as the phage genes. The junction between the III signal sequence and mature III was modified with an NcoI restriction site embedded in the last three codons of the signal sequence. After the III signal, MANP contained the sequence of BPTI with restriction sites for the enzymes StyI, XhoI, PflMI, ApaI, Bsp120I, EcoO109I, PspOMI, BssHII, StuI, BstZ17I, BlpI, EagI, and SphI, which were all unique within MANP. Following BPTI, a unique PstI site and a Factor Xa cleavage site were inserted in a short linker region before mature III. The DNA encoding the library was synthesized with constant DNA on either side so that the DNA could be PCR amplified using TAQ DNA polymerase, cleaved with NcoI and PstI, and ligated to similarly cleaved vector. The variegated parts were synthesized with TRIM technology, which incorporates trinucleotides and allows mixtures of any set of amino acid types in any desired proportions. The flanking and varied DNA sequence positions of TN6-6 phage library is depicted as "AEGTGS-X1X2X2 CX2X2X2X2C X2X2X1-APGPTDS", where X1=ADFGHLNPQRSVWY and X2=ADEFGHIKLMNPQRSTVWY. 340 TN12-1 phage display library 18 Dyax Corp Semi-random Circular The disulfide-constrained cyclic peptide phage display library TN12-1 was constructed in a derivative of M13mp18, MANP, having the following modifications: bla (AmpR) gene, modified junction between signal of iii and coding region for mature III, and removal of LacZ complementation system. MANP comprises 8164 bases. The bla gene was from pGEM3Zf1(+), was bound by BamHI-HindIII sites at the 5' end and HindIII-SalI sites at the 3' end, replaced bases 6001 through 6432 of M13mp18, and was in the same orientation as the phage genes. The junction between the III signal sequence and mature III was modified with an NcoI restriction site embedded in the last three codons of the signal sequence. After the III signal, MANP contained the sequence of BPTI with restriction sites for the enzymes StyI, XhoI, PflMI, ApaI, Bsp120I, EcoO109I, PspOMI, BssHII, StuI, BstZ17I, BlpI, EagI, and SphI, which were all unique within MANP. Following BPTI, a unique PstI site and a Factor Xa cleavage site were inserted in a short linker region before mature III. The DNA encoding the library was synthesized with constant DNA on either side so that the DNA could be PCR amplified using TAQ DNA polymerase, cleaved with NcoI and PstI, and ligated to similarly cleaved vector. The variegated parts were synthesized with TRIM technology, which incorporates trinucleotides and allows mixtures of any set of amino acid types in any desired proportions. The flanking and varied DNA sequence positions of TN6-6 phage library is depicted as "AEGTGD-X1X1X2 CX2X2X2X2X2X2X2X2X2X2C X2X1X1-APGPTDN", where X1=ADFGHLNPRSWY and X2=ADEFGHIKLMNPQRSTVWY. 341 fUSE5-based X10 phage display library 10 2.0e7 B. H. Lindqvist Completely random NNK Linear 342 XCX8CX+X15+X8CX8+X30 M13 phage display library pool 12, 15, 17, 30 Claude Granier Semi-random NNK Linear 343 CX7C T7 phage display library 7 In-San Kim Completely random Circular 344 CX8C phage display library 8 Kristiina Aalto Completely random Circular 345 X3 T7 phage display library 3 Shunsuke Kamijo Completely random Linear The phage display library is based on T7 415-1b phage vector. 346 X4 T7 phage display library 4 Shunsuke Kamijo Completely random Linear The phage display library is based on T7 415-1b phage vector. 347 fUSE5 phage display library Erkki Ruoslahti Semi-random 348 X10-ALLRY-X10 phage display library 25 Igor Fisch Semi-random NNK Linear Illustrate the use of a self-splicing group I intron with inserted lox-Cre recombination site to assemble a very large combinatorial repertoire of peptides from two different exons. Each exon comprised a repertoire of 10 random amino acids residues; after splicing, the repertoires were joined together through a central five-residue spacer to give a combinatorial repertoire of 25-residue peptides. The repertoire was displayed on filamentous bacteriophage fd by fusion to the pIII phage coat protein and selected by binding to several proteins, including β-glucuronidase. 349 X6 fdMED1 phage display library 6 Jean-Pierre Mach Completely random NNK Linear The vector fdMED1 was used for the library construction. In order to avoid the background production of wild-type M13 phage during the selections, a new polylinker cloning site containing several stop codons was constructed between the two unique restriction sites ApaLI and NotI of the original fdDOG1 vector. The construction of this new vector (fdMED1) was performed as follows: the peptide leader sequence (LpelB) of the vector backbone pHEN1 was amplified by PCR with Taq polymerase (Perkin Elmer) using oligo-1 and oligo-fdSEQ01FOR. The reaction mixture (50 mL) was cycled 30 times. The resulting DNA fragment was subcloned into ApaLI and NotI digested fdDOG1 and used to transform Escherichia coli TG1. Clones containing the correct insert were identified by DNA sequencing. In addition, a multiple cloning site (MCS) was generated by inserting annealed oligonucleotides (oligo-4 and oligo-5) into SfiI/NotI digested vector. The DNA was electroporated in E. coli MC1061, thus generating a new fd vector called fdMED1. 350 CX6C fdMED1 phage display library 6 Jean-Pierre Mach Completely random NNK Circular The vector fdMED1 was used for the library construction. In order to avoid the background production of wild-type M13 phage during the selections, a new polylinker cloning site containing several stop codons was constructed between the two unique restriction sites ApaLI and NotI of the original fdDOG1 vector. The construction of this new vector (fdMED1) was performed as follows: the peptide leader sequence (LpelB) of the vector backbone pHEN1 was amplified by PCR with Taq polymerase (Perkin Elmer) using oligo-1 and oligo-fdSEQ01FOR. The reaction mixture (50 mL) was cycled 30 times. The resulting DNA fragment was subcloned into ApaLI and NotI digested fdDOG1 and used to transform Escherichia coli TG1. Clones containing the correct insert were identified by DNA sequencing. In addition, a multiple cloning site (MCS) was generated by inserting annealed oligonucleotides (oligo-4 and oligo-5) into SfiI/NotI digested vector. The DNA was electroporated in E. coli MC1061, thus generating a new fd vector called fdMED1. 351 TN6 phage display library 12 Dyax Corp Semi-random Circular The disulfide-constrained cyclic peptide phage display library TN6 was constructed in a derivative of M13mp18, MANP, having the following modifications: bla (AmpR) gene, modified junction between signal of iii and coding region for mature III, and removal of LacZ complementation system. MANP comprises 8164 bases. The bla gene was from pGEM3Zf1(+), was bound by BamHI-HindIII sites at the 5' end and HindIII-SalI sites at the 3' end, replaced bases 6001 through 6432 of M13mp18, and was in the same orientation as the phage genes. The junction between the III signal sequence and mature III was modified with an NcoI restriction site embedded in the last three codons of the signal sequence. After the III signal, MANP contained the sequence of BPTI with restriction sites for the enzymes StyI, XhoI, PflMI, ApaI, Bsp120I, EcoO109I, PspOMI, BssHII, StuI, BstZ17I, BlpI, EagI, and SphI, which were all unique within MANP. Following BPTI, a unique PstI site and a Factor Xa cleavage site were inserted in a short linker region before mature III. The DNA encoding the library was synthesized with constant DNA on either side so that the DNA could be PCR amplified using TAQ DNA polymerase, cleaved with NcoI and PstI, and ligated to similarly cleaved vector. The variegated parts were synthesized with TRIM technology, which incorporates trinucleotides and allows mixtures of any set of amino acid types in any desired proportions. The varied DNA sequence positions of TN6 phage library is depicted as "X2X1X1 CX1X1X1X1C X1X1X2", where X1=ADFGHLNPQRSVWY and X2=ADEFGHIKLMNPQRSTVWY. 352 TN7 phage display library 11 Dyax Corp Semi-random Circular The disulfide-constrained cyclic peptide phage display library TN6 was constructed in a derivative of M13mp18, MANP, having the following modifications: bla (AmpR) gene, modified junction between signal of iii and coding region for mature III, and removal of LacZ complementation system. MANP comprises 8164 bases. The bla gene was from pGEM3Zf1(+), was bound by BamHI-HindIII sites at the 5' end and HindIII-SalI sites at the 3' end, replaced bases 6001 through 6432 of M13mp18, and was in the same orientation as the phage genes. The junction between the III signal sequence and mature III was modified with an NcoI restriction site embedded in the last three codons of the signal sequence. After the III signal, MANP contained the sequence of BPTI with restriction sites for the enzymes StyI, XhoI, PflMI, ApaI, Bsp120I, EcoO109I, PspOMI, BssHII, StuI, BstZ17I, BlpI, EagI, and SphI, which were all unique within MANP. Following BPTI, a unique PstI site and a Factor Xa cleavage site were inserted in a short linker region before mature III. The DNA encoding the library was synthesized with constant DNA on either side so that the DNA could be PCR amplified using TAQ DNA polymerase, cleaved with NcoI and PstI, and ligated to similarly cleaved vector. The variegated parts were synthesized with TRIM technology, which incorporates trinucleotides and allows mixtures of any set of amino acid types in any desired proportions. The varied DNA sequence positions of TN7 phage library is depicted as "X4X5 CX3X3X3C X5X4", where X3=ADEFGHILNPQRSTVWY, X4=ADFHLPR and X5=ADFGHLPRS. 353 TN8 phage display library 14 Dyax Corp Semi-random Circular The disulfide-constrained cyclic peptide phage display library TN6 was constructed in a derivative of M13mp18, MANP, having the following modifications: bla (AmpR) gene, modified junction between signal of iii and coding region for mature III, and removal of LacZ complementation system. MANP comprises 8164 bases. The bla gene was from pGEM3Zf1(+), was bound by BamHI-HindIII sites at the 5' end and HindIII-SalI sites at the 3' end, replaced bases 6001 through 6432 of M13mp18, and was in the same orientation as the phage genes. The junction between the III signal sequence and mature III was modified with an NcoI restriction site embedded in the last three codons of the signal sequence. After the III signal, MANP contained the sequence of BPTI with restriction sites for the enzymes StyI, XhoI, PflMI, ApaI, Bsp120I, EcoO109I, PspOMI, BssHII, StuI, BstZ17I, BlpI, EagI, and SphI, which were all unique within MANP. Following BPTI, a unique PstI site and a Factor Xa cleavage site were inserted in a short linker region before mature III. The DNA encoding the library was synthesized with constant DNA on either side so that the DNA could be PCR amplified using TAQ DNA polymerase, cleaved with NcoI and PstI, and ligated to similarly cleaved vector. The variegated parts were synthesized with TRIM technology, which incorporates trinucleotides and allows mixtures of any set of amino acid types in any desired proportions. The varied DNA sequence positions of TN8 phage library is depicted as "X6X5X5 CX7X7X7X7X7X7C X5X5X6", where X5=ADFGHLPRS, X6=ADHR and X7=ADFGHLNPQRSVW. 354 TN9 phage display library 11 Dyax Corp Semi-random Circular The disulfide-constrained cyclic peptide phage display library TN6 was constructed in a derivative of M13mp18, MANP, having the following modifications: bla (AmpR) gene, modified junction between signal of iii and coding region for mature III, and removal of LacZ complementation system. MANP comprises 8164 bases. The bla gene was from pGEM3Zf1(+), was bound by BamHI-HindIII sites at the 5' end and HindIII-SalI sites at the 3' end, replaced bases 6001 through 6432 of M13mp18, and was in the same orientation as the phage genes. The junction between the III signal sequence and mature III was modified with an NcoI restriction site embedded in the last three codons of the signal sequence. After the III signal, MANP contained the sequence of BPTI with restriction sites for the enzymes StyI, XhoI, PflMI, ApaI, Bsp120I, EcoO109I, PspOMI, BssHII, StuI, BstZ17I, BlpI, EagI, and SphI, which were all unique within MANP. Following BPTI, a unique PstI site and a Factor Xa cleavage site were inserted in a short linker region before mature III. The DNA encoding the library was synthesized with constant DNA on either side so that the DNA could be PCR amplified using TAQ DNA polymerase, cleaved with NcoI and PstI, and ligated to similarly cleaved vector. The variegated parts were synthesized with TRIM technology, which incorporates trinucleotides and allows mixtures of any set of amino acid types in any desired proportions. The varied DNA sequence positions of TN9 phage library is depicted as "X5 CX7X7X7X7X7X7X7C X5", where X5=ADFGHLPRS and X7=ADFGHLNPQRSVW. 355 TN10 phage display library 16 Dyax Corp Semi-random Circular The disulfide-constrained cyclic peptide phage display library TN6 was constructed in a derivative of M13mp18, MANP, having the following modifications: bla (AmpR) gene, modified junction between signal of iii and coding region for mature III, and removal of LacZ complementation system. MANP comprises 8164 bases. The bla gene was from pGEM3Zf1(+), was bound by BamHI-HindIII sites at the 5' end and HindIII-SalI sites at the 3' end, replaced bases 6001 through 6432 of M13mp18, and was in the same orientation as the phage genes. The junction between the III signal sequence and mature III was modified with an NcoI restriction site embedded in the last three codons of the signal sequence. After the III signal, MANP contained the sequence of BPTI with restriction sites for the enzymes StyI, XhoI, PflMI, ApaI, Bsp120I, EcoO109I, PspOMI, BssHII, StuI, BstZ17I, BlpI, EagI, and SphI, which were all unique within MANP. Following BPTI, a unique PstI site and a Factor Xa cleavage site were inserted in a short linker region before mature III. The DNA encoding the library was synthesized with constant DNA on either side so that the DNA could be PCR amplified using TAQ DNA polymerase, cleaved with NcoI and PstI, and ligated to similarly cleaved vector. The variegated parts were synthesized with TRIM technology, which incorporates trinucleotides and allows mixtures of any set of amino acid types in any desired proportions. The varied DNA sequence positions of TN10 phage library is depicted as "X8X8X2 CX1X1X1X1X1X1X1X1C X2X8X8", where X1=ADFGHLNPQRSVWY, X2=ADEFGHIKLMNPQRSTVWY and X8=DFHLNPRSWY. 356 CX8C fUSE5 phage display library 8 5.0e8 Piero Pollesello (Orion Pharma, R&D) Completely random NNK Circular Briefly, the RF form of the fUSE5 vector of fd phage was prepared by transfecting fUSE5 DNA into E. coli K802 cells. The inserts were prepared by synthesizing degenerate 82-bases-long DNA oligonucleotides containing the sequence: 5\'-CTATTCTCACTCGGCCGACGGGGCTTGCNNT⁄GNNT⁄GNNT⁄GNNT⁄GNNT⁄GNNT⁄GNNT⁄GNNT⁄GTGCGGGGCCGCTGGGGCCGAAACTGTTGAA-3\', and they were used as target molecules in the following PCR reactions. These target sequences were amplified by PCR using synthetic DNA oligonucleotides: 5\'-CTATTCTCACTCGGCCGACG-3\' (5\'-end primer) and 5\'-TTCAACAGTTTCGGCCCCAG-3\' (3\'-end primer). 357 Linear-lib M13 phage display library 8, 10, 12, 14, 16 1.8e11 Rami N Hannoush (Department of Early Discovery Biochemistry, Genentech, South San Francisco, California, USA) Completely random NNK Linear The phage-displayed peptide libraries were constructed by fusing randomized peptides to the N-terminus of M13 major coat protein following the standard protocol for making phage displayed library. 358 Cyclic-lib M13 phage display library 16 7.8e11 Rami N Hannoush (Department of Early Discovery Biochemistry, Genentech, South San Francisco, California, USA) Semi-random NNK Circular The phage-displayed peptide libraries were constructed by fusing randomized peptides to the N-terminus of M13 major coat protein following the standard protocol for making phage displayed library. 359 X4CX10CX4 fUSE5 phage display library 20 David N. Krag Semi-random NNK Circular The library was constructed using the fUSE5 gene III phage-display system. 360 X2CX4CX FUSE5-based phage display library 7 1e12 Erkki Ruoslahti (Research Center, The Burnham Institute, California) Semi-random NNK Circular The library was constructed in the vector FUSE5. 361 X7 FUSE5-based phage display library 7 1e12 Completely random NNK Linear The library was constructed in the vector FUSE5. 362 SX8 phage display library (SX8) 8 1.8e8 2e13 Kay Completely random NNK Linear The library was constructed using a novel peptide phage display vector mJ1. mJ1 was constructed by inserting the AmpR gene into the EcoRI site of mBAX27, a derivative of M13mp18. Using the poly-merase chain reaction (PCR), the pIII signal sequence cleavage site and N terminus of pIII were replaced with a new cloning site composed of a XhoI site and BamHI site flanking an amber stop codon. Recombinants have a single serine residue fixed at the N terminus of the mature pIII protein to ensure minimal bias during signal sequence cleavage, and a (Gly-Ser)4 spacer between the peptide and full-length pIII protein. 363 CMTI phage display library 9 2.0e7 Dyax,Cambridge,MA Semi-random Circular The CMTI phage display library consists of M13 E.coli bacteriophage carrying a 90-bp (30aa) insert into the amino terminus of the pⅢ coat protein consisiting of 23 constant amino acids and seven variable amino acid residues. 364 LX15 phage display library 15 G. Smith , University of Missouri, Colombia, MO Completely random Circular 365 X3CX7-10CX3 T7 phage display library Semi-random NNK Circular The T7 phage libraries displaying typically X3CX7-10CX3 random peptides, where X represents the randomized amino acid positions generated using mixed oligonucleotides on template DNA, were constructed using the T7 Select vector 10-3b. 366 X7-LXXLL-X7 T7 phage display library 19 1.3e7 2.6e11 Novagen Semi-random Linear For construction of the X7-LXXLL-X7 phage library, complementary DNA strand of ON165F was generated using extension primer ON166R and klenow fragment. The DNA fragments were digested by EcoRI and HindIII and the resulting EcoRI-HindIII restriction fragment was cloned into T7Select 10-3b EcoR I/HindIII Vector Arms, which allow 5-15 copies of peptides to be randomly displayed on the phage surface. 367 X15 phage display library 15 K. D. Janda Completely random NNK Linear The phage-display vector pCGMT-1b was used as a template for the generation of the peptide-pVII and peptide-pIX fusion gene repertoires. 368 X4CX10CX10 FUSE5 phage display 20 1.8e7 Stephen Albert Johnston (Affymax, Palo Alto, CA) Semi-random Circular 369 NSLTPCX7C T7 phage display library 14 1.0e9 Narisorn. N Completely random NNK Circular Each synthesized oligonucleotide was ligated to T7 vector arm. Target peptides were expressed as fusion to the C-terminus of the 10B capsid protein and were displayed on the virion surface. 370 CX9C phage display library 9 Heejoon Myung Completely random NNK Circular The C-9-C mer constrained peptide library was constructed using the pCANTAB5E vector (Pharmacia, U.S.A.). Two oligomers were annealed to encode a C(NNK)9C peptide and extended with a Klenow fragment and dNTPs. The resulting short double-stranded DNA was then ligated between the SfiI and NotI sites. E. coli JM109 was transformed with the ligated DNAs and further infected with helper M13 phages. 371 ANL4 phage display library (X10C) 10 1.6e10 Brian K. Kay Completely random Circular The library is based on M13TAG phage. 372 ANL5 phage display library (X8CX2) 11 2.1e10 Brian K. Kay Semi-random Circular The library is based on M13TAG phage. 373 CX6C FUSE5-based phage display library 6 1.2e8 Robert O. Fox Completely random NNK Circular The library of DNA fragments was cloned into the SfiI sites of the Fuse-5 vector and transfected into Escherichia colistrain DH5α. 374 X7 T7 phage display library 7 Y.sagane Completely random Linear Heptapeptide library was constructed using T7Select 415-1. 375 X9 PC89-based phage display library 9 9.4e7 Franco F Completely random NNN Linear The library was based on PC89 vector. Random nucleotide sequences were inserted into the region encoding the amino terminus of pa 376 SUT12 M13 phage display library 12 M. Yamabhai(Suranaree University of Technology, Thailand) Completely random NNK Linear The phage display library of a random peptide was constructed by cloning DNA inserts assembled from synthetic degenerate oligonucleotides (NN(G/T)12) into an M13 vector, such that the random peptides were expressed as N-terminal fusions to the M13 minor coat protein pIII. The complexity of the library is ∼10e9 members. The titer of the library is ∼10e10 pfu. 377 Ph.D.-12 and Ph.D.-7 phage library pool 7, 12 New England BioLabs, Beverly, MA, USA Completely random NNK Linear 378 X7 and X11 FUSE5 phage display library pool 7, 11 Karyon-CTT Ltd Completely random Linear 379 f88-4 phage display library pool 1.0e8~1.0e9 G. Smith (University of Missouri, Columbia, MO), Bonnycastle Semi-random The peptides were displayed as fusions with the pⅧ coat protein of the f88-4 phage display vector. X6, X15, X30, LX4(XCX4CX), LX6(XCX6CX), LX8(XCX8CX), LX10(XCX10CX), LX12(XCX12CX), Cys3(X5CX3CX4), Cys4(X4CX4CX4), Cys5(X4CX5CX4), Cys6(X4CX6CX4), X8CX8, X15CX and XCCX3CX5C4GIEGRG library were included in the phage display library pool. 380 Ph.D.-7 and Ph.D.-C7C phage display library pool 7 New England BioLabs, Beverly, MA, USA Completely random NNK 381 f5-15mer phage display library (X15) 15 G. Smith (University of Missouri, Columbia, MO) Completely random NNM Linear The phage library was constructed based on the vector fUSE5. It expressed 15-mer random peptides near the N-terminus of phage surface protein pIII. 382 fd-tet X8 phage display library 8 Chuanbin Mao Completely random Linear The landscape phage library contains billions of fd-tet phage clones and in each phage clone the foreign peptide is displayed on the N-terminal end of each copy of major coat protein of fd-tet phage. 383 M13 pVIII phage display library e11 Gregory A. Weiss Semi-random NNS Random peptide sequences CX5C, X2CX4CX2, X4CX10CX4, CX5CX, X2CX5CX2, X5CX8CX5, CX5CX2, X2CX6CX2, X5CX9CX4, XCX5C, X2CX7CX2, X6CX6CX6, X2CX5C, X2CX8CX2, X6CX7CX5, X2CX9CX2, X7CX4CX7, X2CX2CX2, X2CX10CX2, X7CX5CX6, X2CX2CX3, X2CX3CX2, X4CX2GPX4CX4 and X8 were in the phage display library. Random peptide segments (containing both linear and disulfide crosslinks) were expressed on the major M13 coat protein pVIII using a two-plasmid phagemid/helper phage system. 384 Ph.D.-12 phage display and wild type library pool Completely random The phage display 12-mer library is doped with 10% wild type library (i.e. phage that do not express any sequence on their surfaces). 385 T7 X6 phage display library 6 1.5e8 Navneet Sharma Completely random NNK Linear The sequence of synthetic degenerate oligonucleotides inserted in the coding region of T7 phage capsid protein (employing T7 Select 1-1 vector arms, T7 Select system, Novagen, Canada), encoding a random hexamer followed by (His)6 tag is as follows: 59-AAT TCT CTC ACT CCA GGC GGC-(NNK)6-GGT GGT CAT CAC CAT CAC CAT CAC TAA-39 (N represents any nucleotide and K represents T or G). 386 pComb8 CX15C phage display library 17 Department of Biochemistry, University of Amsterdam (Amsterdam, The Netherlands) Completely random Circular 387 pIF15 phage display library 15 Dr. Paolo Monaci (Istituto di Ricerca di Biologia Molecolare, Rome, Italy) Completely random Linear 388 pⅧ and peptides-on-plasmids phage display library pool Semi-random NNK Phagemid libraries displaying random peptides fused to the NH2-terminus of pⅧ were of the form X10, X11, X20, GGCX8C, GGCX10C, and GGCX12C. Libraries displaying random peptides fused to the COOH-terminus of the lac repressor protein were of the form X15, X4CX(4-10)CX4 and X3CX(5-10)CX3. 389 pVIII-9aa.Cys phage display library (CX9C) 11 Dr. A. Nicosia (Institute of Molecular Biology, Pomezia, Rome) Completely random Circular 390 T7 CX9C phage display library 11 Richard G. Smith (University of Nottingham, Cancer Research Laboratories, School of Pharmaceutical Sciences, Nottingham NG7 2RD, UK) Semi-random NNK Circular The T7Select Phage Display System immunology utilizing the T7Select415-1b vector (Novagen, Inc., Madison, WI, USA), was used to display approximately 415 identical copies of each peptide on the capsid head. The peptides were encoded for by a degenerate oligonucleotide insert, consisting of a randomized library of 9-residue peptides flanked by cysteine residues (to impose a structural constraint on the C-terminus of the displayed peptides). These cysteine were also flanked with serine residues, such that the N-terminus of the peptide linked to the capsid head through a serine residue. 391 CX(7-11)C phage display library 7-11 Cwirla SE (Department of Molecular Biology, Affymax Research Institute, Palo Alto, CA, USA.) Completely random NNK Circular The library was constructed on the phagemid vector p8V2. This is a disulfide constrained library expressing peptides of 7-11 random amino acid peptides flanked by two cysteine residues and linked to the N terminus of the major pVIII phage coat protein. 392 Linear-lib and Cyclic-lib M13 phage display library pool 8, 10, 12, 14, 16 Rami N Hannoush (Department of Early Discovery Biochemistry, Genentech, South San Francisco, California, USA) Semi-random NNK Linear Two groups of phage-displayed peptide libraries, the linear peptide library called Linear-lib and cysteine-restrained cyclic library called Cyclic-lib, were constructed by fusing randomized peptides to the N terminus of M13 major coat protein p8. Linear-lib consisted of random peptides with 8, 10, 12, 14, or 16 amino acids, and Cyclic-lib consisted of 14-mer random peptides with varied length between two invariant cysteines. The final diversities for Linear-lib and Cyclic-Lib were 1.8e11 and 7.8e11, respectively. 393 SGTACX2GPX4CSLAGSP phage display library 20 Sidhu SS (Department of Protein Engineering, Genentech, Inc., South San Francisco, California) Semi-random Circular The library consisted of e8-e109 independent transformants. It was displayed on gene 8, under the control of the Ptac promoter. 394 X4CX2GPX4CX4 phage display library 18 Sidhu SS (Department of Protein Engineering, Genentech, Inc., South San Francisco, California) Semi-random Circular The library consisted of e8-e109 independent transformants. It was displayed on gene 8, under the control of the Ptac promoter. 395 X5CX8CX5 phage display library 20 Sidhu SS (Department of Protein Engineering, Genentech, Inc., South San Francisco, California) Semi-random Circular 396 X2CX6CX2 phage display library 12 Sidhu SS (Department of Protein Engineering, Genentech, Inc., South San Francisco, California) Semi-random Circular 397 X5CX9CX4 phage display library 20 Sidhu SS (Department of Protein Engineering, Genentech, Inc., South San Francisco, California) Semi-random Circular 398 X2CX3CX2 phage display library 9 Sidhu SS (Department of Protein Engineering, Genentech, Inc., South San Francisco, California) Semi-random Circular 399 X2CX8CX2 phage display library 14 Sidhu SS (Department of Protein Engineering, Genentech, Inc., South San Francisco, California) Semi-random Circular 400 X2CX10CX2 phage display library 16 Sidhu SS (Department of Protein Engineering, Genentech, Inc., South San Francisco, California) Semi-random Circular 401 X20 FUSE5-based phage display library 20 5.0e8 Baruch Stern (Department of Cell Research and Immunology, George S Wise Faculty of Life Science, Tel-Aviv University, Israel) Completely random NNK Linear The library was constructed by cloning a 60-nucleotide-long random sequence (correspondmg to a 20-amino acid random sequence) into the pa 402 f88-4/Cys6 phage display library (X4CX6CX4) 16 G. Smith (University of Missouri, Columbia, MO) Semi-random NNM Circular The f88-cys6 is a 16 amino acid sequence randomized for every residue except for the locations of two cysteines and containing more than 2.7e8 phage. 403 T7 PXTGTWX8G phage display library 8 Watanabe H and Yamasaki K (The Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology) Semi-random NNK Linear The library was generated by elongating a segment containing an eight-residue randomized region and a glycine residue Xaa8-Gly at the C terminus of a chignolin-derived segment termed CLN. Here, the Xaa was encoded by a degenerate codon NNK. The gene fragments were synthesized by overlap extension polymerase chain reaction (PCR), digested by restriction enzymes EcoRI/HindIII, and then ligated into the C-terminal part of a T7 phage coat protein gene 10 (g10) of the T7Select 10 -3b vector (Novagen). 404 X28 and X28 M13 phage display library pool 28 Completely random NNK Linear Two libraries displayed 28 amino acid-long random peptides as fusions to the major capsid protein VIII of M13. 405 Twelve different M13 phage libraries pool Completely random NNK Linear 406 X20 phage display library 20 Pillutla RC and Hsiao K (DGI BioTechnologies, Inc, Edison NJ, USA) Completely random DNA fragments coding for peptides containing 20 random amino acids were generated by a PCR approach using synthetic oligonucleotides. Peptides are expressed on the capsid protein pIII of the phage at low copy number (1–2 peptides/phage). 407 X40 phage display library 40 Pillutla RC and Hsiao K (DGI BioTechnologies, Inc, Edison NJ, USA) Completely random DNA fragments coding for peptides containing 40 random amino acids were generated by a PCR approach using synthetic oligonucleotides. Peptides are expressed on the capsid protein pIII of the phage at low copy number (1–2 peptides/phage). 408 fUSE5 X5WYA[WF]SPX4 phage dsiplay library 15 Landon LA (University of Missouri, Columbia, Missouri, USA) Semi-random NNK Linear The plus-strand sequence was 5'-TCG GCC GAC GGG GCC (NNK)5 TGG TAT GCG TGG TCC CCG (NNK)4 GGG GCC TCT GGG GCC GAA AGT-3'. The library is based on FUSE5 vector. Random peptides were express on protein III. The resulting peptides contained the six-amino-acid TF antigen–binding consensus sequence (W-Y-A-W/F-S-P) in the conserved central part of the peptide and terminal regions of random amino acid sequence. The large size of the input library (1.0e11 bacteriophage particles) indicated that a large percentage of the possible 15-amino-acid peptides were represented in the library. 409 CX8C T7 phage display library 8 5.0e8 Pilch J (Burnham Institute for Medical Research, USA) Completely random NNK Circular The cyclic peptide library with the general structure of CX8C was based on a T7 10–3b phage vector. 410 T9 M13 phage display library (X22) 22 2.0e8 Brian K. Kay Completely random NNK Linear The library contains 2e8 unique clones expressing 22-amino acid-long random peptides fused to the amino terminus of M13 protein Ⅲ. 411 T12 M13 phage display library (X36) 36 2.0e8 Brian K. Kay Completely random NNK Linear The library contains 2e8 unique clones expressing 36-amino acid-long random peptides fused to the amino terminus of M13 protein Ⅲ. 412 X6PX2PX6 M13 phage display library 16 3.0e8 Brian K. Kay Semi-random NNK Linear Peptides were expressed as N-terminal fusions to the mature protein Ⅲ of Bacteriophage M13. 413 X12 M13 phage display ibrary 12 Brian K. Kay Completely random NNK Linear The library was based on M13 phage. 414 f3-7mer fUSE5-based phage display library 7 G. Smith (University of Missouri, Columbia, MO) Completely random NNK Linear Library f3-7mer is based on vector fUSE5, which displys its guest peptides on the minor coat protein pIII at one tip of the virion. 415 X8CX8 f88-based phage display library 17 2.5e8 J. K. Scott (Simon Fraser University, Burnaby, Canada) Semi-random NNK Linear The library is based on vector f88, which displays its guest peptides on the coat protein pVIII. 416 X4CX4CX4 f88-4-based phage display library 14 G. Smith (University of Missouri, Columbia, MO) Semi-random NNK Circular The peptides were displayed as fusions with the pⅧ coat protein of the f88-4 phage display vector. 417 pComb3.5 XSX5 phage display library 7 Barbas CF 3rd Semi-random NNK Linear The murine Cys2-His2 zinc finger protein Zif268 was used for construction of phage-display libraries. Primer pairs Aseq (5'-GTCCATAAGATTAGCGGATCC-3') and Zfl6rb [5'-CTGGCCTGTGTGGATGCGGATATG(MNN)5CGAMNNAGAAAAGCGGCGATCGCAGGA-3', where N is A, T, G, or C and M is A or C], and Bseq (5'-GTGAGCGAGGAAGCGGAAGAG-3') and Zflf (5'-CATATCCGCATCCACACAGGCCAG-3') were used to amplify fragments of the Zif268 gene using plasmid pAra-Zif268 as a template. The two PCR fragments were used as templates for overlap extension, and the library was constructed in pComb3.5. 418 pComb3.5 X6 phage display library 6 Barbas CF 3rd Completely random NNK Linear The murine Cys2-His2 zinc finger protein Zif268 was used for construction of phage-display libraries. Primer pairs Aseq (5'-GTCCATAAGATTAGCGGATCC-3') and Zff2r6f [5'-CAGTGTCGAATATGCATGCGTAACTTC(NNK)6ACCACCCACATCCGCACCCAC-3', where N is A, T, G, or C and K is G or T], and Bseq (5'-GTGAGCGAGGAAGCGGAAGAG-3') and Zfnsilb (5'-CATGCATATTCGACACTGGAA-3') were used to amplify fragments of the Zif268 gene using plasmid pAra-Zif268 as a template. The two PCR fragments were used as templates for overlap extension, and the library was constructed in pComb3.5. 419 pComb3H SX4LX2 phage display library 8 4.4e9 Barbas CF 3rd Semi-random NNK Linear The zinc finger library was based on vector pComb3H. It involved randomization of residues within the a-helix of finger 2 of C7, a variant of Zif268. The NNK library was constructed by randomization of positions -1, 1, 2, 3, 5, and 6 by using a condon doping strategy that allows for all amino acid combinations within 32 condons. The position -2 is somewhat artifactual; the NNK library had this residue fixed as serine. Also it contained an invariant leucine at position 4. 420 pComb3H X5LX2 phage display library 8 3.5e9 Semi-random Linear The zinc finger library is based on vector pComb3H. It involved randomization of residues within the a-helix of finger 2 of C7, a variant of Zif268. The library was constructed by randomization of positions -2, -1, 1, 2, 3, 5, and 6, which precludes Tyr, Phe, Cys, and all stop condons in its 24-codon set and contained an invariant leucine at position 4. 421 X10 phagemid-based fd phage display library 10 6.0e9 Completely random NNK Linear Random peptides were fused to the NH2-terminus of pⅧ. 422 X12 phagemid-based fd phage display library 12 2.0e8 Completely random NNK Linear Random peptides were fused to the NH2-terminus of pⅧ. 423 CX8C phagemid-based fd phage display library 8 2.0e9 Completely random NNK Circular Random peptides were fused to the NH2-terminus of pⅧ. 424 pZif12 X15 phage display library 15 Carl O. Pabo (Massachusetts Institute of Technology, Department of Biology, Cambridge, USA) Completely random Linear Phagemid vectors used in the selections were created from pZif12 by restoring the reading frame between the Zif12-coding region and gene III and by introducing convenient restriction sites at the start of Zif12. The library containing randomized 15-residue peptides was constructed by cassette mutagenesis, using NN(G/C/T) randomized codons. The complete fusion protein used for phage display contained a PelB signal sequence; a short leader peptide (NH2-EPRAQNS); the random peptide; residues 4–60 of Zif268; a linker that includes an amber codon; and residues 23–424 of M13 gene III product. 425 XnCXnCXn and Xn M13 phage display library pool ~4.7e11 Gregory A. Weiss (University of California, Irvine, California) Semi-random NNK Linear The phage display library pool was composed of 15 different scaffolds that feature different arrangements of conformation biasing disulfides and randomized residues. XnCXnCXn and Xn type libraries fused to the P8 major coat protein of M13 phage were constructed. 426 fGWX10 phage display library 10 Kenneth H. Pearce Completely random NNK Linear A modified polyvalent phage display vector, fGWg3, was constructed using the original vector fTC. The library is based on fGWg3 and displays a random 10-residue peptide sequence with flanking regions as follows: NH2-EDGGSX10(GGGGS)3-gIII protein. 427 X7LXX[HI]IXXX[IL]X7 CoRNR box phage display library 23 1.3e7 Semi-random NNK Linear The 23-mer phage library in which the CoRNR box motif was flanked on each side by seven random amino acid residues (X7-L-X-X-H/I-I-X-X-X-I/L-X7) was created in the phage vector mBAX such that the recombinant peptide is expressed as a fusion with the pIII capsid protein in an M13 bacteriophage. 428 SSX6PPX6SR M13 phage display library 14 1.5e9 1.0e13 Brian K . Kay and Marius Sudol Semi-random NNK Linear The library is based on a bacteriophage M13 display vector with the reading frame of the insert defined by restriction sites within gene III. The insert corresponds to the general sequence: SSX6PPX6SR, encoded by TCGAGC(NNK)6CCACCT(NNK)6TCTAGA where N = G+A+T+C, K = G+T, and X= all 20 amino acids. 429 CX12C phage display library 12 Suk-Jung Choi Completely random NNK Circular The linearized pCANTAB5E was ligated with oligonucleotides. Random oligonucleotides are placed between the gene III signal sequence and the E-tag. The E-tag peptide facilitates the identification and purification of a displayed peptide. The randomized sequence of the inserted oligonucleotide consists of twelve NNK triplets where N=A, C, G, or T, and K=G or T. This will produce a fusion protein consisting of a random peptide, E-tag, and gene III protein. The helper phage M13K07 was used to produce a phage library from the bacteria harboring library phagemid. 430 CX5C+CX6C+CX9 FUSE5-based phage display library pool 5, 6, 9 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Completely random NNK Circular Peptide libraries were constructed on the FUSE5 vector. 431 CX5C+CX6C+CX7C FUSE5 phage display library pool 5, 6, 7 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Completely random NNK Circular Peptide libraries were constructed on the FUSE5 vector. 432 X10ALLRYX10 phage display library 25 Igor Fisch Semi-random NNK Linear The repertoire of 25 residue "spliced" peptides displayed on phage was created by in vivo recombination between phage and plasmid replicons, followed by self-splicing of the RNA between the two exons (exon 1: NNK10GCTCTCT and exon 2: TAAGGTATNNK10). Each exon comprised a repertoire of 10 random amino acids residues; after splicing, the repertoires were joined together through a central five-residue spacer to give a combinatorial repertoire of 25-residue peptides. The repertoire was displayed on filamentous bacteriophage by fusion to the pIII phage coat protein. 433 X6PNDKYEPF phage display library 6 1.0e7 John Doorbar Completely random NNK Linear As the terminal six residues of the tethered ligand appear to have binding activity, this region was rondomised, but eight residues of the tether were retained. The tethered ligand library was prepared in the vector fdtetSfi/Not following digestion with SfiⅠ and NotⅠ. The oligonucleotide used for library preparation is 5'-G TTG TTC CTT TCT ATG CGG CCC CAG CCG GCC ATG GCA (NN(G/T))6 CCG AAC GAT AAG TAC GAG CCG TTC CCA CCA CCA CCA GCG GCC GCA GAA ACT GTT CGC GCG CGC GAA CAG-3'. The displayed sequences were linked to gene Ⅲ protein by a rigid poly-proline spacee. 434 R26 M13 phage display library (SRX12(A/S/P/T)A(A/D/E/G/V)X12SR) 26 2.0e8 Semi-random NNK Linear The library was constructed by annealing and extending two long degenerate oligos with a complementary region at their 3' termini. The 6-nt complementarity corresponded to the SacII recognition sequence and encoded the tripeptide: (A/S/P/T)A(A/D/E/G/V). This design, which fixed Ala as the central aa, permits the subdividing of the long peptides for the analysis of binding residues by SacII digestion of the DNA insert. The library consisted of 2e8 recombinants, each expressing the peptide sequence: SRX12(A/S/P/T)A(A/D/E/G/V)X12SR at the mature N-terminus of pIII. 435 X6 M13 phage display library 6 6.4e7 Jacek Otlewski (University of Wroctaw, Wroctaw, Poland) Completely random NNS Linear The phage-displayed library of random hexapeptides was fused to the C terminus of the M13 phage major coat protein P8, downstream of the AWEENIDSAP linker to ensure optimal C-terminal display of peptide variants on the phage surface. The insert was obtained by hybridization of degenerate and phosphorylated oligonucleotides: bib(for) TCGAGCGGTNSNNSNNSNNSNNSNNSTAATAA and bib(rev) CTAGTTATTASNNSNNSNNSNNSNNSNNACCGC (where N stands for equimolar contribution of A, T, G, C and S for G or C). The resulting cassette with sticky ends (XhoI/SpeI) was inserted into the modified phagemid pComb3H. 436 fUSE5-based CX7C phage display library 7 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Completely random NNK Circular The phage library displaying CX7C was constructed on fUSE5 vector. It was designed to display a constrained cyclic loop within the pIII capsid protein. 437 X7+X12+CX7C M13 phage display library pool 7, 12 New England Biolabs Completely random NNK Circular 438 X7 phage display library 7 1.0e11 Completely random Linear 439 f88.4-based X15 phage display library 15 J. Scott (Simon Fraser University, Burnaby BC, Canada) Completely random Linear The 15-mer linear peptides were displayed at the N-terminus of the pVIII protein of the filamentous phage f88.4. 440 XCX3CX3CX phage display library 11 5.6e8 Christian Heinis, Institute of Chemical Sciences and Engineering, Switzerland Semi-random NNK Circular The library was cloned as follows. The genes encoding semi-random peptides with the sequence Xaa-Cys-(Xaa)3-Cys-(Xaa)3-Cys-Xaa, the linker Ser-His-Ser and the two disulfidefree domains D1 and D2 were cloned in the correct orientation into the phage vector 21tet. The vector is based on the phage vector fdg3p0ss21 and contains a 2.5 kb stuffer fragment instead of the gene region coding for the D1 and D2 domains of phage p3. 441 XCX4CX4CX phage display library 13 4e8 Christian Heinis, Institute of Chemical Sciences and Engineering, Switzerland Semi-random NNK Circular The library was cloned as follows. The genes encoding semi-random peptides with the sequence Xaa-Cys-(Xaa)4-Cys-(Xaa)4-Cys-Xaa, the linker Ser-His-Ser and the two disulfide-free domains D1 and D2 were cloned in the correct orientation into the phage vector fd0ssD12. 442 CX6CX6C phage display library 15 4e9 Christian Heinis, Institute of Chemical Sciences and Engineering, Switzerland Semi-random NNK Circular The library was cloned as follows. The genes encoding semi-random peptides with the sequence Cys-(Xaa)6-Cys-(Xaa)6-Cys, the linker Ser-His-Ser and the two disulfide-free domains D1 and D2 were cloned in the correct orientation into the phage vector fd0ssD12. 443 CX3CX3C phage display library 9 Christian Heinis, Institute of Chemical Sciences and Engineering, Switzerland Semi-random NNK Circular The library was cloned as follows. The genes encoding semi-random peptides with the sequence Cys-(Xaa)3-Cys-(Xaa)3-Cys, the linker Ser-His-Ser and the two disulfide-free domains D1 and D2 were cloned in the correct orientation into the phage vector fd0ssD12. 444 CX5CX5C phage display library 13 Christian Heinis, Institute of Chemical Sciences and Engineering, Switzerland Semi-random NNK Circular The library was cloned as follows. The genes encoding semi-random peptides with the sequence Cys-(Xaa)5-Cys-(Xaa)5-Cys, the linker Ser-His-Ser and the two disulfide-free domains D1 and D2 were cloned in the correct orientation into the phage vector fd0ssD12. 445 Acid-pp M13 phage display library (XSAX2KX) 7 2.22e8 Freie Universität Berlin Semi-random NNK Linear The library sequences were expressed as pIII protein fusions on M13 bacterophage surface. 446 CX8C M13 phage display library 8 3.4e13 Completely random NNK Circular The library was made by ligating a synthetic 42-bp fragment into the M13KE vector and transfecting E. coli cells with the ligation product by electroporation. Within the fragment was the degenerate coding sequence 5′-GCTTGT (NNK)8 TGCGGTGGAGGT-3′, where N stands for an equimolar mixture of G, A, T, and C while K is an equimolar mixture of G and T. The single strand degenerate oligonucleotide is converted to double strand by polymerase chain reaction (PCR) using the extension primer 5′-CATGCCCGGGTACCTTTCTATTCTC-3′ (New England Biolabs Inc., Ipswich, MA). 447 X8 M13 phage display library 8 Completely random NNK Linear The library was made by ligating a synthetic 33-bp fragment into the M13KE vector and transfecting E. coli cells with the ligation product by electroporation. Within the fragment was the degenerate coding sequence 5′-(NNK)8GGTGGAGGT-3', where N stands for an equimolar mixture of G, A, T, and C, while K is an equimolar mixture of G and T. The single strand degenerate oligonucleotide is converted to double strand by polymerase chain reaction (PCR) using the extension primer 5'-CATGCCCGGGTACCTTTCTATTCTC-3'. 448 FliTrx M13 phage display library (X7) 7 New England Biolabs Completely random NNK Linear FliTrx phage display library (X7) was from New England Biolabs. 449 CX8C fUSE5 phage display library 8 ~8.0e9 Fabiano Pinheiro da Silva (University of Sao Paulo, Sao Paulo, Brazil) Completely random Circular The phage library displaying CX8C peptides (C, cysteine; X, any amino acid) was constructed using the vector fUSE55. 450 CX7C T7 phage display library 7 Completely random NNK Circular 451 X2CX6CX2 M13 phage display library Semi-random NNK Circular A cyclic M13 phagemid libraries were constructed using standard molecular methods and transfected in Escherichia coli TG1 cells using a MicroPulser Electroporator (Bio-Rad). 452 X12 M13 phage display library Completely random NNK Linear A linear M13 phagemid libraries were constructed using standard molecular methods and transfected in Escherichia coli TG1 cells using a MicroPulser Electroporator (Bio-Rad). 453 TATA-cyclized CXnCXnC phage display library (CXnCXnC) 9/11/15 Christian Heinis (Institute of Chemical Sciences and Engineering, Switzerland) Semi-random NNK Circular The DNA coding for the random peptide sequences is added to the gene coding for domains D1 and D2 of the cysteine free pIII by PCR using a degenerate primer. The DNA coding for the peptide-D1–D2 fusion is then introduced into the fd-based vector fd0D12 between a leader sequence and the third domain(D3) of pIII. The libraries were generated by cyclizing linear peptides of the format CXnCXnC (C = cysteine, X = random amino acid; n= 3, 4, 6) displayed on the phage with the thiol-reactive reagent 1,3,5-triacryloyl-1,3,5-triazinane (TATA). 454 TBAB-cyclized CXnCXnC phage display library (CXnCXnC) 9/11/15 Christian Heinis (Institute of Chemical Sciences and Engineering, Switzerland) Semi-random NNK Circular The DNA coding for the random peptide sequences is added to the gene coding for domains D1 and D2 of the cysteine free pIII by PCR using a degenerate primer. The DNA coding for the peptide-D1–D2 fusion is then introduced into the fd-based vector fd0D12 between a leader sequence and the third domain(D3) of pIII. The libraries were generated by cyclizing linear peptides of the format CXnCXnC (C = cysteine, X = random amino acid; n= 3, 4, 6) displayed on the phage with the thiol-reactive reagent N,N′,N″-(benzene-1,3,5-triyl)-tris(2-bromoacetamide) (TBAB). 455 CX7-10C T7 phage display library 7-10 Kotaro Sakamoto (Pharmaceutical Research Division, Japan) Completely random Circular 456 X13 T7 phage display library 13 Completely random Linear The T7 phage libraries displaying random peptides, which were generated by mixed-oligonucleotides as template DNA, were internally constructed by using T7Select 10-3 vector from Merck Millipore (Darmstadt, Germany) 457 F88-FUSE X4CX4CX4 phage display library (X4CX4CX4) 14 Creative Biolabs, Shirley, NY Semi-random NNK Circular The phage library was constucted on the phage vector f88.4. 458 X5CX10CX T7 phage display library (X5CX10CX) 18 Kotaro Sakamoto (Pharmaceutical Research Division, Japan) Semi-random Circular 459 Bacteriophage MS2 VLP DENV-3 antigen fragment library 10 Kathryn M. Frietze (University of New Mexico, USA) Completely random We produce libraries of peptides on MS2 VLPs by site±directed mutagenesis of MS2 coat protein in pDSP62 after the method of Kunkel.The library was constructed by synthesizing overlapping 30 nucleotide oligonucleotides corresponding to the DENV-3 genome using a massively parallel microchip-based synthesis method. After amplification by PCR, the oligonucleotides were then used to construct a library of VLP expression plasmids encoding every possible 10-mer DENV-3 peptide. The library was then expressed as VLPs in E. coli and used in subsequent deep sequencecoupled biopanning experiments. 460 X15 fUSE55 phage display library 15 6.6e7 Sangdun Choi (Ajou University, Suwon, South Korea) Completely random NNM Linear A 15-mer peptide library was synthesized with forward primer 5'-TTG ATC GCA AGG ATC GGC TAG C-3' and reverse primer 5' -AA GGC CTT GGT ACC GCT GCC ACC (MNN)15 GCT AGC CGA TCC TTG CGA TCA A-3'. These two primers were annealed and then elongated using Pfu DNA polymerase (SolGent, Daejeon, Korea) for 30 min at 68℃. The DNA product was digested withNheI/KpnI and cloned into the fUSE55 vector with T4 DNA ligase (New England Biolabs, Inc., Ipswich, MA, USA). The DNA library was transformed into electrocompetent Escherichia coli(E. coli) DH10B cells, generating 6.6e7 distinct clones. 461 X12 pHEN2 phage display library 12 2.0e9 Sangdun Choi (Ajou University, Suwon, South Korea) Completely random NNK Linear The 12-mer peptide library was synthesized with forward primer 5'-GCC CAG CCG GCC ATG GCC (NNK)12 TCG AGT GGT GGA GGC GGT TCA G-3' and reverse primer 5'-GCC AGC ATT GAC AGG AGG TTG AG-3'. The two primers were annealed and elongated as described above. Then, the DNA product was digested withNcoI/BamHI and cloned into the pHEN2 phagemid vector with T4 DNA ligase. The library was then transformed into electrocompetent E. coli DH10B cells, generating 2.0e9 distinct clones. 462 T7 phage display library Kotaro Sakamoto (Pharmaceutical Research Division, Japan) The T7 phage-displayed random peptide libraries, which were generated by using mixed oligonucleotides as template DNA, were constructed by using a T7Select 10-3 vector from Merck (Darmstadt, Germany). 463 X6 T7 phage display library 6 2.5e6 Kazuhisa Sugimura (Kagoshima University, Korimoto, Japan) Completely random NNM Linear The T7 phage library displaying random 6-mer peptides was constructed using the phage vector T7Select415 (Novagen). To add hexameric amino acid sequences to the C-terminal of the G10 protein of T7 phage through the GGGS linker peptide, a template oligonucleotide (5'-GCCGCAAGCTTTTATCCMNNMNNMNNMNNMNNMNNCGAACCTCCACCTGAATTCGG-3') was synthesized. This oligonucleotide was amplified by PCR using forward and reverse primers (5'-CCGAATTCAGGTGGAGG-3'and 5'-CGGCGTTCGAAAATAGG-3') that harbored restriction sites for EcoRI and HindIII, respectively. The generated PCR product was purified on a PCR-M column (Viogene) and digested with EcoRI and HindIII. The DNA fragment was purified on a PCR-M column again and ligated into the corresponding restriction sites in the T7Select415 vector. The ligation mixture was subjected to anin vitro packaging reaction using a T7 packaging extract (Novagen). The packaged T7 phages were infected into Escherichia coli BL21 and amplified once. 464 X16 and X12 M13 phage display library 12 and 16 Andreas Ernst and Ivan Dikic (Institute of Biochemistry II, Goethe University, Germany) Completely random Linear The 16-mer peptide library with 1.0e12 unique peptides was expressed on M13 coat protein p8. The peptide-coding sequences were assembled from trimeric nucleotide building blocks, so that each naturally occurring amino acid (with the exception of cysteine) was encoded with equal probability at each of the 16 amino acid positions. This approach alleviates the risk of introducing unwanted stop codons. The 12-mer peptide library consisted of 1.0e11 unique peptides fused to M13 minor coat protein p3. The peptide-coding sequence was assembled from 12 consecutive repetitions of “NNK” codons (where N represents all four nucleotides and K represents guanine or thymine). 465 X16 phagemid library 16 Holger Spiegel(Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany) Linear The trinucleotide-based, 16-mer peptide phagemid library covered >1.0e10 clones (ENTE-1). 466 pVIII fth1-dp-based X7 phage display library (X7) 7 Jonathan M. Gershoni (Tel Aviv University, Tel Aviv, Israel) Completely random NNK Linear The fth1-dp vector is a derivative of the fth1 vector. To adapt the fth1 for Illumina deep sequencing, Illumina adaptors A and B were inserted upstream and downstream to the insert-flanking SfiI sites. Oligonucleotides corresponding to the Adaptor sequences were inserted by 'SOEing' PCR mutagenesis using the Accuzyme polymerase (Bioline, BIO-21052). Alternatively, extended PCR primers can be used to accomplish the same as has been described previously. Libraries were constructed as described below. Briefly, two 5’ biotinylated oligonucleotides were used. The first contained the redundant "library" sequence, e.g., 7×NNK flanked by BglI sites compatible with the two SfiI sites of the vector (61 bases). The second oligonucleotide, 18 bases, complemented the 3’ end of the first and was extended to "fill-in" the complementary strand using Klenow polymerase. The product was digested with BglI, the short biotinylated segments were removed with Streptavidin conjugated magnetic beads and the eluent was cloned into SfiI digested fth1-dp vector. This ligation mix was used to electroporate MC1061 or DH5alpha cells as indicated in the text. 467 CX3CX4CG, CX4CX3CG, CX4CX4CG, CX3CX5CG and CX5CX3CG phage display library pool 10, 11 Christian Heinis, Institute of Chemical Sciences and Engineering, Switzerland Semi-random NNK Circular Peptides are displayed on around five copies of the phage coat protein pIII. The peptide library pool contain peptides of the format ACXmCXnCG (C = cysteine, X = any amino acid). The combinations of ‘m’ and ‘n’ are 3/4, 4/3, 4/4, 3/5 and 5/3. The genes encoding semi-random peptides, the linker Ser-His-Ser and the two disulfide free domains D1 and D2 were cloned in the correct orientation into the phage vector 21tet. 468 CX3CX6CG, CX6CX3CG, CX4CX5CG and CX5CX4CG phage display library pool 12 Christian Heinis, Institute of Chemical Sciences and Engineering, Switzerland Semi-random NNK Circular Peptides are displayed on around five copies of the phage coat protein pIII. The peptide library pool contain peptides of the format ACXmCXnCG (C = cysteine, X = any amino acid). The combinations of ‘m’ and ‘n’ are 3/6, 6/3, 4/5 and 5/4. The genes encoding semi-random peptides, the linker Ser-His-Ser and the two disulfide free domains D1 and D2 were cloned in the correct orientation into the phage vector 21tet. 469 XCX3CX3CX and XCX4CX4CX phage display library pool 11, 13 Christian Heinis, Institute of Chemical Sciences and Engineering, Switzerland Semi-random NNK Circular The library was cloned as follows. The genes encoding semi-random peptides, the linker Ser-His-Ser and the two disulfidefree domains D1 and D2 were cloned in the correct orientation into the phage vector 21tet. 470 Ph.D.-5 M13 phage display library (X5) 5 Naoto Oku (University of Shizuoka, Japan) Completely random NNK Linear A phage-displayed random peptide library expressing pentapeptides at the N terminus of gIII coat protein of M13 phage was constructed by using the Ph.D. Peptide Display Cloning System (New England Biolabs, Beverly, MA, USA). 471 Aptide pIGT2-based phage display library (X6GSWTWENGKWTWKGX6) 26 8e8 Sangyong Jon (Gwangju Institute of Science and Technology, Gwangju, South Korea) Semi-random NNK Circular An aptide comprises a stabilizing scaffold and two target-binding regions. The scaffold consists of a small (12 amino acids) but highly stable tryptophan zipper that forms a leucine-zipper-like b-hairpin structure, in which two tryptophan–tryptophan cross-strand pairs create a robust and stable structure. To mimic the DNA recognition site of bZIP proteins two target-binding regions, each comprising six randomizable amino acids, are introduced at both ends of the trpzip scaffold through glycine linkers. To create a large and highly diverse aptide library, we synthesized two degenerate aptide-encoding oligonucleotides: aptide-F1, 5'-TTC TAT GCG GCC CAG CTG GCC (NNK)6 GGA TCT TGG ACA TGG GAA AAC GGA AAA-3 and aptide-B1, 5’-AAC AGT TTC TGC GGC CGC TCC TCC TCC (MNN)6 TCC CTT CCA TGT CCA TTT TCC GTT-3’, where N was A, T, G, or C; K was G or T; and M was C or A (Genotech). A double-stranded randomized insert was created using polymerase chain reaction (PCR), double-digested with SfiI/NotI (New England Biolabs) and cloned into SfiI/NotI-digested pIGT2 phagemid vectors (Ig Therapy Co.). The resulting construct was electroporated into electrocompetent E. coli XL1 cells (Stratagene), and the transformed cells were infected with Ex12 mutant helper phage (Ig Therapy Co.) for amplification of the aptide-displaying phage library. 472 pVIII AE(X)2DP phage display library (AEX2DP) 2 1.02e3 Junho Chun (Seoul National University College of Medicine,Seoul,Republic of Korea) Completely random NNK Linear A restriction endonuclease recognition site (KpnI) was introduced into the sequence encoding the pVIII coat protein leader peptide (LV), using the SnaBI and BamHI restriction enzyme sites, which are located in the VCSM13 genome. Two amino acids (GD) exposed at the N-terminal pVIII coat protein were replaced with random amino acids encoded by NNK degenerate codons (N =A, T,G and C,and K=G and T). 473 pVIII AE(X)3DP phage display library (AEX3DP) 3 3.28e4 Junho Chun (Seoul National University College of Medicine,Seoul,Republic of Korea) Completely random NNK Linear A restriction endonuclease recognition site (KpnI) was introduced into the sequence encoding the pVIII coat protein leader peptide (LV), using the SnaBI and BamHI restriction enzyme sites, which are located in the VCSM13 genome. Two amino acids (GD) exposed at the N-terminal pVIII coat protein were replaced with random amino acids encoded by NNK degenerate codons (N =A, T,G and C,and K=G and T). 474 pVIII AE(X)4DP phage display library (AEX4DP) 4 1.05e6 Junho Chun (Seoul National University College of Medicine,Seoul,Republic of Korea) Completely random NNK Linear A restriction endonuclease recognition site (KpnI) was introduced into the sequence encoding the pVIII coat protein leader peptide (LV), using the SnaBI and BamHI restriction enzyme sites, which are located in the VCSM13 genome. Two amino acids (GD) exposed at the N-terminal pVIII coat protein were replaced with random amino acids encoded by NNK degenerate codons (N =A, T,G and C,and K=G and T). 475 SFTI-based phage display (SFTI8Ph) library (X8) 8 Uwe Haberkorn (German Cancer Research Center,(DKFZ) Heidelberg, Germany) Completely random NNN Linear The combinatorial sunflower trypsin inhibitor 1-based phage display (SFTI8Ph) library based on the sunflower trypsin inhibitor (SFTI) 1 scaffold structure was constructed by PCR. To this end, 8 amino acids except cysteine were randomly inserted between Thr4 and Cys11 in the binding loop of SFTI-1. The following oligonucleotides were used as templates for the PCR reaction: 5‘-TTACTCGCTCCATGGGCGGCAGGTGTACTNNN NNN NNN NNN NNN NNN NNN NNN TGTTATCCCGAT-3’ (SFTI8Ph forward; NNN = trimer encoding for one variable amino acid; Ella Biotech) and 5`-ATAATCTTGCGGCCGCACCGCCACCTGCTGCATCGGGATAACA-3` (SFTIPh reverse; Eurofins MWG Operon). With regard to the 125I-labeling of peptides the triplet TTT was exchanged by TAT to achieve a substitution of phenylalanine 12 to tyrosine. The PCR product was cloned in frame into the surface expression phagemid vector pSEX81 (Progen) and single clones were sequenced (GATC Biotech) to control the diversity of the library. For the preparation of phages presenting the peptides (theoretical diversity of 109) fused to the pIII coat protein on their surface the phagemid vector was transformed in XL1Blue bacteria. 476 X6TLSPKMPGGGYW M13 bacteriophage library (X6TLSPKMPGGGYW) 18 1.00e8 Creative Biolabs, Shirley, NY Completely random NNK Linear Two phage libraries were then constructed (Creative Biolabs, Shirley, NY) in which TMT-2 (TLSPKMPGGGYW) formed the backbone on the M13KE RF vector. To the N-terminus of each bacteriophage 12-mer backbone, six amino acids were added at random using the NNK synthesis strategy, resulting in 1.0e8 distinct 18-mers in each library. 477 X6SADSTKTTHLTL M13 bacteriophage library (X6SADSTKTTHLTL) 18 1.00e8 Creative Biolabs, Shirley, NY Completely random NNK Linear Two phage libraries were then constructed (Creative Biolabs, Shirley, NY) in which TMT-3 (SADSTKTTHLTL) formed the backbone on the M13KE RF vector. To the N-terminus of each bacteriophage 12-mer backbone, six amino acids were added at random using the NNK synthesis strategy, resulting in 1.0e8 distinct 18-mers in each library. 478 T7 X3CX9-10CX3 phage display library (X3CX9-10CX3) 17-18 Yuji Ito (Kagoshima University, Kagoshima, Japan) Semi-random NNK Circular T7 phage libraries typically displaying X3CX9–10CX3 random peptides, where X represents randomized amino acid positions generated using mixed oligonucleotides on template DNA, were constructed using the T7Select vector 10-3b from Merck (Kenilworth, NJ, USA). 479 TAFTSWEEYLDWVGSG fusion M13 phage display library (TAFTSWEEYLDWVGSGX(8,10,12,14,16)) 8, 10, 12, 14, 16 1.3e10 Daniel Kirchhofer (Department of Early Discovery Biochemistry, Genentech, Inc., South San Francisco, California, USA) Semi-random NNK Circular The libraries were constructed using the standard Kunkel mutagenesis method. The Pep2-8V2A fusion peptide libraries were constructed by fusing randomized peptides via a GSG linker to the C terminus of the anchor peptide Pep2-8V2A (TAFTSWEEYLDWVGSG). The fusion peptide libraries were displayed on the N terminus of M13 major coat protein (p8) following the standard protocol for making phage-displayed libraries. The extension pool consisted of random peptides with 8, 10, 12, 14, and 16 amino acids in length encoded by consecutive degenerate codons (NNK, where N = A/C/G/T and K = G/T). The libraries with different lengths were constructed individually and pooled together at equal concentrations. The final diversity of the library was 1.3e10. 480 WNLVRIGLLR fusion peptide library (X(2-5)(G/S)WNLVRIGLLR) 2, 3, 4, 5 2.5e10 Daniel Kirchhofer (Department of Early Discovery Biochemistry, Genentech, Inc., South San Francisco, California, USA) Completely random NNK Linear The WNLVRIGLLR fusion peptide library was constructed by fusing randomized peptides ranging 2–5 residues to the N terminus of peptide (G/S)WNLVRIGLLR. The randomized peptides were encoded by consecutive degenerate codon NNK and glycine/serine by RGT (R = A/G). The fusion peptide libraries were displayed on the N terminus of M13 major coat protein (p8) following the standard protocol for making phage-displayed libraries. The extension pool consisted of random peptides with 2, 3, 4 and 5 amino acids in length. The libraries with different lengths were constructed individually and pooled together at equal concentrations. The final diversity of the library was 2.5e10. 481 f88-Cys5 phage display library (X4CX5CX4) 15 5.90E+08 G. Smith (University of Missouri, Columbia, MO) Semi-random NNK Circular The f88–Cys5 phage display library (GenBank Accession AF246454) is based on vector f88-4 (Accession AF218363). The filamentous phage-display library displaying random peptides on the virion surface, fused to a recombinant version of the major coat protein pVIII. Each peptide has two cysteines at fixed positions 5 residues apart within the otherwise randomized amino acids, and thus is potentially constrained conformationally by a disulfide bond. The random amino acids are specified by degenerate codons in a synthetic oligonucleotide insert, which was spliced into vector f88-4 between its HindIII and PstI cloning sites in recombinant gene rVIII. The resulting recombinant, insert-bearing rVIII-Cys5 gene, encoding recombinant protein rpVIII-Cys5, is inducible by IPTG. The phage genome also contains the wild-type gene VIII, which is expressed constitutively. Most of the 4000 major coat-protein subunits in the virion derive from the wild-type VIII gene, but under fully induced conditions (1 mM IPTG) up to 300 subunits are rpVIII-Cys5 polypeptides derive from the recombinant, insert-bearing rVIII-Cys5 gene. The library comprises 5.9e8 primary phage clones altogether, each represented by many phage particles as a result of replication of the library en masse in Escherichia coli host cells. 482 XS2X9, Ph.D.-12 and wild-type phage library pool 12 Andreas Herrmann (University of Groningen, Groningen, The Netherlands) Semi-random Linear The XS2X9 library, PhD-12 library and wild-type phages were pooled. The different phage pools were mixed in a 1:1:1 ratio. 483 T7 LARFH gene-based phage display library (X3GX2) 6 7.40E+06 Akihiko Yamagishi (Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan) Semi-random Linear To create an LARFH gene library, amino acid residues at positions 21, 22, 23, 25, and 26 were subjected to semi-random mutagenesis. Gly24 was not mutated. Each of their codons was replaced with the NVS codon (N = A + T + G + C,V = A + G + C, S = G + C). Genes were then amplified by using two different primers so that the EcoRI and HindIII recognition sites could be added at the 5' and 3' termini, respectively. Amplified DNA fragments were digested with EcoRI and HindIII and then ligated to T7 Select Vector Arms (Merck Millipore, Germany). For in vitro packaging, ligated DNA fragments were added to T7 phage packaging extracts (Merck Millipore) and the mixtures were incubated at 25 °C for 2 h. After phage packaging, a T7 phage library containing 7.4e6 independent clones was constructed. 484 T7 X12, X16 and X20 phage display library pool 12, 16, 20 Taiji Asami (Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., Fujisawa) Completely random NNK Linear To generate T7 phage libraries displaying random peptides, X12, X16, X20 (X is a mixture of twenty natural amino acids) mixed-oligonucleotides as template DNA were internally constructed, purified with a QIAquick PCR Purification kit (QIAGEN, Hilden, Germany), and ligated into the T7Select 10-3 vector (Merck Millipore, Darmstadt, Germany), according to the manufacturer's manual. The total library diversity was estimated to be 3.1e9 plaque-forming units (pfu). 485 GX12 phage display library 12 H. G. Börner(Department of Chemistry, Humboldt-Universität zu Berlin, Germany) Completely random 486 X6 phage display library 6 >5.5e6 2.5e8 Colin A. Kretz (McMaster University) Completely random NNK Linear The random nucleotide libraries were either inserted into FUSE67, or designed to contain a FLAG-tag 5′ to the variable region before cloning into the FUSE55 phage display vector. 487 VWF73(P3-P3′) phage display library(X6) 6 2.5e7 Colin A. Kretz (McMaster University) Completely random NNK Linear Random 6 amino acid peptide libraries were also constructed in the context of VWF73 (Asp1596-Arg1668 of VWF), replacing the codons for Leu8-Thr13 with the degenerate codon series, NNK. The P3-P3′ residues within VWF73 were replaced with random amino acids. 488 X7FTSDYSKYLDSRRAQDFVQWLX2T, X7SDYSKYLDSRRAQDFVQWLX2T, HSQGTFX7LDSRRAQDFVQWLX2T, HSQGTFTSDYSKYX7DFVQWLX2T and HSQGTFTSDYSKYLDSRRAQX9 phage display library pool 31,29,29,29,29 6e7 Antonello Pessi(PeptiPharma, Roma, Italy) Semi-random NNK Linear The Phage display library used to select GCGR/GLP1R co-agonists is composed of five sub-libraries, each one with nine randomized positions, indicated with a X letter. The sub-libraries were constructed on the pCANTAB6 phagemid vector. Because of the chosen randomization scheme, each peptide displays 1–3 mutations distributed across the 9 randomized positions, with the remaining positions featuring the wt residue. For selection on GCGR+ and GLP1R+ cells, the five libraries were pulled (‘Library Mix’) and selected together. 489 IX104 phage display library(X5CX8CX5) 20 1.40e10 Sepideh Afshar(Department of protein Engineering, Eli Lilly Biotechnology Center, California) Semi-random NNK Peptide phage libraries were generated using the IX104 bacteriophage vector. Escherichia coli strain RZ1032 (ATCC 39737) was used to prepare uracil containing single-stranded DNA (du-ssDNA) of the IX104 vector. A library oligonucleotide, containing the random amino acid sequences encoded by NNK was designed such that the random NNK region was flanked by nucleotides complementary to the vector. 5'-phosphorylated reverse complement oligo was annealed to dUssDNA IX104 vector using Kunkel mutagenesis and extended to form double stranded DNA (dsDNA). 490 IX104 phage display library(X3CX12CX3) 20 3.7e10 Sepideh Afshar(Department of protein Engineering, Eli Lilly Biotechnology Center, California) Semi-random NNK Peptide phage libraries were generated using the IX104 bacteriophage vector. Escherichia coli strain RZ1032 (ATCC 39737) was used to prepare uracil containing single-stranded DNA (du-ssDNA) of the IX104 vector. A library oligonucleotide, containing the random amino acid sequences encoded by NNK was designed such that the random NNK region was flanked by nucleotides complementary to the vector. 5'-phosphorylated reverse complement oligo was annealed to dUssDNA IX104 vector using Kunkel mutagenesis and extended to form double stranded DNA (dsDNA). 491 X9 T7 phage display library 9 5e7 Lars Hellman(Uppsala University, Sweden) Completely random NNK Linear The general sequence of the T7 phage capsid proteins are PGG(X)9HHHHHH, where (X)9 indicates the randomized nonamers. Each phage clone expresses a unique sequence of 9 random amino acids (nonamer) on their surface, followed by a His6-tag in the C-terminus of capsid protein 10. 492 CXCX5CX5C M13 phage display library 15 4.5e9 1.8e10 Chuanliu Wu(Xiamen University, China) Semi-random NNK Circular A library of peptides comprising two sequences of five random amino acids flanked by a CXC motif and two isolated cysteines for display on the M13 phages (CXC(X)5C(X)5C, where X is any amino acid encoded by NNK) was designed. The random peptide is linked via a triple alanine (Ala–Ala–Ala) linker to the gene-3 protein (pIII). A glycine residue was added to the N-terminus of the random peptide to ensure the removal of the signal sequence. Transformation of the phagemid vector into E. coli yielded a library of 4.5e9 independent tranformants, and around 1.8e10 infective phages can be produced per ml of culture. 493 CGPX12GPC bacterial display library (CGPX12GPC) 12 Thermo Fischer Scientific, MA, USA Completely random Circular The bacterial peptide display library was composed of 280 million collections of random dodecamer peptides that were displayed on the N-term end of flagellin in bacterial cell membranes. 494 TriCo-16™ phage display peptide library (X16) 16 1.0e9 Creative Biolabs Completely random NNK Linear The phage library was constructed on the M13 phage vector. 495 SGPI-2 XCX4CX M13 phage display library 6 7.0e8 Gábor Pál (ELTE Eötvös Loránd University, Hungary) Completely random NNK The SGPI-2 library was based on the Tag-SGPI-2-pGP8 phagemid vector. The P4, P2, P1, P1, P2' and P4’positions were fully randomized, while the disulfide-bonded P3 and P3' Cys residues were kept as wild-type. 496 SPINK1 GCX6 M13 phage display library 6 6.25e8 Gábor Pál (ELTE Eötvös Loránd University, Hungary) Completely random NNK The synthetic gene encoding human SPINK1 (UniProt: P00995) was purchased from Genscript and was subcloned into a phagemid vector to be fused to the M13 p8 coat protein gene. Then, six codons corresponding to binding loop positions P2-P4’were replaced with stop codons by Kunkel mutagenesis. Next, in a combinatorial mutagenesis step, the stop codons were replaced by NNK codons encoding all 20 amino acids to produce the SPINK1-phagemid library. 497 CX6CX6C phage display library 15 8.8e7 Christoph Ernst(Eberhard Karls Universität Tübingen, Tübingen, Germany) Semi-random Linear The display vector used for the library is fd-phage fd0D12. The phage library with a complexity of 8.8e7 different clones presenting linear peptides of the format ACX6CX6CG-phage (X = all natural occurring amino acids except cysteine) was prepared as follows: 30 μg SfiI digested fd0D12 vector was ligated with 9 μg (3-fold molar excess) of the respective library insert. 498 XDXXFNXINXAXXVXXVNXXKNX phage display library 23 4e15 Jakeb M. Reis (University of Toronto, Toronto, Canada) Semi-random Linear The libraries were prepared by site-directed mutagenesis. The following oligonucleotides were used for mutagenesis of randomized positions. GA domain: 5’-AAGGCTGGTATCACC(N1)(N2)(K1)GAC(N1)(N2)(K1)(N1)(N2)(K1)TTCAAC(N1)(N2)(K1)ATCAAT(N1)(N2)(K1)GCG(N1)(N2)(K1)(N1)(N2)(K1)GTG(N1)(N2)(K1)(N1)(N2)(K1)GTTAAC(N1)(N2)(K1)(N1)(N2)(K1)AAGAAC(N1)(N2)(K1) ATCCTGAAAGCTCAC -3’, where (N1) is a custom mix of 10% A, 20% C, 20% G and 50% T, (N2) is a custom mix of 40% A, 25% C, 10% G and 25% T, (K1) is a custom mix of 30% G and 70% T. The scaffold library (GA domain) was displayed fused to the M13 coat protein pVIII using a custom 8+8 type phagemid. 503 TN2 phage display library (XCX4CX) 8 8.55e6 Protein Engineering Corporation (Cambridge, MA 02138, USA) Semi-random Circular The M13-derived vector, called MKTN, was utilized. MKTN contains unique AccIII and CelII sites adjacent to the DNA encoding the SP processing site of III, and a KmR gene within the intergenic region. The TN2 phage display library was constructed by ligating variegated DNA into MKTN RF DNA cut with AccIII and CelII. 504 fUSE55-based X6 phage display library 6 Ricardo J. Giordano (Universidade de São Paulo, São Paulo, Brazil) Completely random NNK Linear The linear hexapeptide phage display (X6) library was built as previously described but with modifications. Briefly, the fUSE55 vector (provided by G. Smith, University of Missouri, Columbia, MO) was prepared in large scale using the Maxiprep kit (Qiagen) followed by two consecutive CsCl equilibrium gradient purifications. Equimolar amounts of oligonucleotides 5′-CACTCGGCCGACGGGGCTNNKNNKNNKNNKNNKNNKGGGGCCGCTGGGGCCGAA- 3′ and 5′- TTCGGCCCCAGCGGC-3′ (where N = any nucleotide and K = T or G) were converted to double-stranded DNA with Klenow enzyme (as recommended by the manufacturer) (New England Biolabs) and purified using a P500 Maxiprep column (Qiagen). The vector (500 mg) and oligonucleotide insert (20 mg) digested with the restriction enzyme Bg lI were ligated using T4 DNA ligase (New England Biolabs). The ligation product was purified using a P500 Maxiprep column and transformed into electrocompetent Escherichia coli MC1061 cells, resulting in 1.4e9 transformants, of which 1.2e9 contained inserts coding for peptides. Bacteria were cultured for ~20 hours, and phages were purified fromculture supernatants by the polyethylene glycol/NaCl method. 505 fUSE55-based CX8C phage display library (CX8C) 8 Ricardo J. Giordanoa (University of São Paulo, São Paulo, Brazil) Completely random NNK Circular Briefly, the fUSE55 vector was prepared in large scale with the Maxiprep kit (Qiagen) followed by two consecutive CsCl equilibrium gradient purifications. Equimolar amounts of oligonucleotides 5′- CACTCGGCCGACGGGGCTTGCNNKNNKNNKNNKNNKNNKNNKNNKTGCGGGGCCGCTGGGGCCGAA-3′ and 5′-TTCGGCCCCAGCGGC-3′ (where N = any nucleotide and K = T or G) were converted to double-stranded DNA with Klenow enzyme (as per the manufacturer, New England Biolabs) and purified on a P500 Maxiprep column (Qiagen). The vector (1 mg) and oligonucleotide insert (40 μg) digested with the restriction enzyme BglI were ligated with T4 DNA ligase (New England Biolabs), and the product was purified in a P500 Maxiprep column and transformed into electrocompetent Escherichia coli (MC1061 strain) cells. Bacteria were cultured in Luria-Bertani (LB) broth media supplemented with streptomycin (25 μg/ml) and tetracyclin (20 μg/ml) for ~20 hours at 37°C and 200 rpm, and phage were purified from culture supernatants by the polyethylene glycol/NaCl method (PEG/NaCl). 506 X7CX7CX7 M13 phage display library (X7CX7CX7) 23 Bradley T. Messmer (Abreos Biosciences, San Diego, CA, USA) Semi-random Circular Each displayed peptide contained a C-terminal GGG linker as the fusion point of the displayed peptide and the N-terminus of the pIII M13 phage coat protein. 507 ENTE-1 phage display library (GX3[C/S]X12) 16 Michael Szardenings (Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany) Semi-random Linear The vector pPepPr3A-stuffer was used to generate the phage display library ENTE-1. 508 TN7/1 phage display library 11 1.1e8 Dyax Corp Semi-random Circular The TN7/1 phage display library was constructed in the previously described M13mp18 phage derivative, MANP. The variegated parts were synthesized with TRIM technology, which incorporates trinucleotides and allows mixtures of any set of amino acid types in any desired proportions. The varied DNA sequence positions of TN7/1 phage library is depicted as "X1X2CX3X3X3X3X3CX2X1", where X1 = ADFHLPR, X2 = ADFGHLPRS, X3 = ADEFGHILNPQRSTVWY. The use of TRIM allows addition of preformed trinucleotides to the growing synthetic DNA to express the desired specification of amino acid residue distributions at each varied position. The library DNA was purified, and ligated between the NcoI and PstI sites in the prepared MANP vector. The ligated DNA products were used to transform cells of the Escherichia coli strain XL1-Blue (Stratagene). 509 TN8/6 phage display library 12 1.7e8 Dyax Corp Semi-random Circular The TN8/6 phage library was constructed in the previously described M13mp18 phage derivative, MANP. The variegated parts were synthesized with TRIM technology, which incorporates trinucleotides and allows mixtures of any set of amino acid types in any desired proportions. The varied DNA sequence positions of TN8/6 phage library is depicted as "X4X2CX5X5X5X5X5X5CX2X4", where X4 = ADHR, X2 = ADFGHLPRS, X5 = ADFGHLNPQRSVW. The use of TRIM allows addition of preformed trinucleotides to the growing synthetic DNA to express the desired specification of amino acid residue distributions at each varied position. The library DNA was purified, and ligated between the NcoI and PstI sites in the prepared MANP vector. The ligated DNA products were used to transform cells of the Escherichia coli strain XL1-Blue (Stratagene). 510 CX7C T7 phage display library 7 Byungheon Lee (Kyungpook National University, Daegu, Republic of Korea) Circular During synthesis of oligonucleotides encoding the seven amino acids, those encoding hydrophobic amino acids were enriched to occur at a frequency of at least one residue in every seven residues. A T7 phage vector was purchased from Merck Millipore (Burlington, MA) and genetically engineered to display the CX7C peptides at the carboxy-terminal end of the phage coat protein. 511 T7 SCX8CS phage display library (SCX8CS) 8 9.88E+7 4.87E+11 Nobuhiro Ishida (TOYOTA Central R&D Labs, Inc., Nagakute, Japan) NNK Circular The library displaying random peptides was constructed using a T7 Select 10-3b system (Merck Millipore, Burlington, MA, USA). 512 T7 CX7C phage display library (CX7C) 7 Merk Circular 513 T7 X7 phage display library (X7) 7 NNM Linear A T7 phage library displaying random 7‐mer peptides was constructed using the phage vector T7Select 415 (Merck). Briefly, the template oligonucleotide (5′‐GCCGCAAGCTTTTATCCMNNMNNMNNMNNMNNMNNMNNACCTCCACC TGAATTCGG ATC‐3′) was synthesized to add random amino acid sequences to the C‐terminus of the G10 protein of T7 phage via the triple glycine (GGG) as a linker peptide, and it was amplified by PCR using forward and reverse primers (5′‐GATCCGAATTCAGGTGGAGG‐3′ and 5′‐GCCGC AAGCTTTTATCC‐3′, respectively), which included the restriction enzyme sites for EcoRI and HindIII, respectively. DNA fragments obtained by PCR were digested with EcoRI and HindIII and were ligated into the same restriction sites in the T7Select 415 vector. The in vitro packaging was performed using a T7 packaging extract (Merck) according to the manufacturer's protocol, and the packaged T7 phages were used to infect Escherichia coli BL21 cells and amplified once. 514 CX7C T7 phage display library (CX7C) 7 370000 1.24e11 Debadyuti Ghosha (University of Texas, Austin, USA) Completely random NNK Circular A combinatorial library of cyclic heptapeptides flanked by a pair of cysteine residues displayed on T7Select415-1 phage (Novagen, WI; catalog number 70015) was constructed according to the manufacturer's protocol. Briefly, degenerate NNK-oligonucleotides (Supplementary Table 1) encoding a library of heptapeptides with the general structure CX7C were synthesized and obtained from Integrated DNA Technologies (IDT, IL). To create the library insert, oligonucleotides (primers 1 and 2, Supplementary Table 1) were annealed, extended with DNA polymerase I Klenow fragment (NEB, MA; catalog number M0210S), double digested with HindIII-HF/EcoRI-HF (NEB, MA; catalog numbers R3104S and R3101S), and cloned into the T7Select 415-1 HindIII/EcoRI vector arms (Novagen, WI product number RC0135) to enable peptide display on the 415 copies of the T7 gp10A phage capsid protein as a C-terminal fusion. Next, the peptide-displayed phage library was amplified once in liquid culture with BL21 E. coli (Novagen, WI; product number RC0131). The resulting lysate was clarified by centrifugation at 8000 ×g and stored long-term in 0.1 volume sterile 80% glycerol at −80 °C. 515 pADLg3-TGC-(NNK)6-TAG phagemid library 6 Wenshe Ray Liu (Texas A&M University, College Station, USA) Completely random NNK Circular To afford a phagemid library for the production of phages with displayed cyclic peptides, we inserted a 24 base-pair DNA fragment that encoded six randomized amino acids flanked by an N-terminal cysteine and a C-terminal AcrK between the PelB leader peptide-coding region and the gIII gene of the pADLg3 phagemid. Briefly, we constructed the phagemid library by undergoing PCR to directly amplify the pADL-10b plasmid using two primers pADL-F: 5'- GGTCCGTCCA TGGCCTGCNN KNNKNNKNNK NNKNNKTAGG GCCCGGG-3', and pADL-R: 5'-CCACGGCCAT GGCCGGCTG GGCCGCG-3'. We digested the PCR product using the NcoI restriction enzyme and ligated the digested product using T4 DNA ligase. DpnI was also used to remove the template phagemid. We then electroporated the ligated plasmids into competent E. coli Top10 cells, incubated the transformants in 1 mL LB medium, and then inoculated it into 50 mL LB medium containing 100 μg/mL ampicillin. After OD600 reached 1.0, we collected 0.5 mL of the cell culture, mixed it with 50% glycerol, and stored in -80 °C. 516 pVIII-12aa phage display library (X12) 12 Franco Felici (Istituto di Ricerche di Biologia Molecolare, Rome, Italy) Completely random NNN Linear The phage M13 library was used, kindly donated by Prof. Franco Felici, expressing random peptides exposed on the pVIII protein, based on the phagemid vector pC8970 in which random oligonucleotide sequences were inserted in the region 5′ of the VIII gene present in the vector, under the control of the LacZ promoter. The digestion with the restriction enzymes EcoRI and BamHI linearized the vector and allowed the insertion of the oligonucleotides with random sequences, which flanked by the same restriction sites allow the recircularization of the vector through ligase reaction. 517 pVIII-12aa-Cys phage display library (X3CX4CX3) 12 Franco Felici (Istituto di Ricerche di Biologia Molecolare, Rome, Italy) Completely random NNN Circular The phage M13 library was used, kindly donated by Prof. Franco Felici, expressing random peptides exposed on the pVIII protein, based on the phagemid vector pC8970 in which random oligonucleotide sequences were inserted in the region 5′ of the VIII gene present in the vector, under the control of the LacZ promoter. The digestion with the restriction enzymes EcoRI and BamHI linearized the vector and allowed the insertion of the oligonucleotides with random sequences, which flanked by the same restriction sites allow the recircularization of the vector through ligase reaction. 518 pIT2 X2ELX2LIX2 phage display library 10 5e7 Sara Linse (Biochemistry and Structural Biology, Lund University, SE-22100 Lund, Sweden) Linear The library was cloned in frame with gene III in the pIT2 phagemid vector for display at the N terminus of protein 3. The KM13 helper phage is used to rescue the SXkmer/pIII fusion that preferentially packages the single-stranded phagemid DNA in the presence of the phagemid wild-type M13 origin of replication. The use of the phagemid/helper phage also permits monovalent phage display for the selection of higher-affinity binders. DNA codons were chosen to yield the following amino acid sequences of the displayed proteins: SXkmer - side: MKSPEELKRIFEKYAAKEGDPDQLSXXELXXLIXXEFPSLLKGMSTLDDLFQELDKDGDGEVSFEEFQVLVKKISQ where X denotes any amino acid except cysteine. The theoretical number of variants is 5e7. The DNA synthesis and cloning into the pIT2 vector was purchased from Twist Biosciences. 519 pIT2 GGGX7GGG phage display library 10 9e8 Sara Linse (Biochemistry and Structural Biology, Lund University, SE-22100 Lund, Sweden) Linear The library was cloned in frame with gene III in the pIT2 phagemid vector for display at the N terminus of protein 3. The KM13 helper phage is used to rescue the SXkmer/pIII fusion that preferentially packages the single-stranded phagemid DNA in the presence of the phagemid wild-type M13 origin of replication. The use of the phagemid/helper phage also permits monovalent phage display for the selection of higher-affinity binders. DNA codons were chosen to yield the following amino acid sequences of the displayed proteins: SXKmer - loop: MKSPEELKRIFEKYAAKEGDPDQLSKDELKLLIQAEFPSLLKGMGGGXXXXXXXGGGSTLDDLFQELDKDGDGEVSFEEFQVLVKKISQ, where X denotes any amino acid except cysteine. The theoretical number of variants is 9e8. The DNA synthesis and cloning into the pIT2 vector was purchased from Twist Biosciences. 520 X9 phage display library 9 Shanghai Ray Gene Biotechnology Co., Ltd (Shanghai, China) The phage random 9 peptide library was purchased from Shanghai Ray Gene Biotechnology Co., Ltd (Shanghai, China). 521 CX9C phage display library 11 Sandip M. Kanse (Oslo University Hospital and Medical Faculty, University of Oslo, 0372 Oslo, Norway) A library template phagemid was prepared by inserting a short oligo containing two consecutive stop codons (ocher/opal) into the NcoI/BamHI expression cassette of the pGALD9ΔL phagemid using standard methods. Using this template, a Cys-constrained random 9-mer (NNKC9) peptide library fused to pIX was constructed based on Kunkel mutagenesis essentially as described. The final E. coli SS320 transformation frequency-based library sizes were about 1.0e10 unique clones, and the libraries were prepared with high-valence peptide display by rescue with DeltaPhage. 522 X11 phage display library 11 Sandip M. Kanse (Oslo University Hospital and Medical Faculty, University of Oslo, 0372 Oslo, Norway) Linear A library template phagemid was prepared by inserting a short oligo containing two consecutive stop codons (ocher/opal) into the NcoI/BamHI expression cassette of the pGALD9ΔL phagemid using standard methods. Using this template, a linear random 11-mer (NNK11) peptide library fused to pIX was constructed based on Kunkel mutagenesis essentially as described. The final E. coli SS320 transformation frequency-based library sizes were about 1.0e10 unique clones, and the library was prepared with high-valence peptide display by rescue with DeltaPhage. 523 CX7C and CX8C phage display library pool 9-10 Erkki Koivunen (Division of Biochemistry, University of Helsinki, Helsinki, Finland) Circular 524 fUSE5-based CX8C phage display library 10 1.0e9 Wadih Arap (Rutgers Cancer Institute of New Jersey, Newark, USA) Circular 525 AXXXXXXXGGS phage display library 11 Shunsuke Tagami(RIKEN Center for Biosystems Dynamics Research, Tsurumi-ku, Japan) Linear We first prepared the peptide library with seven randomized amino acid residues (AXXXXXXXGGS) fused to pIII of the pHEN1 phagemid vector. The phagemid library was then expressed and purified by PEG precipitation. 526 Novagen T7 X7 phage display library 7 William C. Sessa1(Vascular Biology and Therapeutics Program and Department of Pharmacology, Yale University School of Medicine, New Haven, USA) Linear Novagen T7 select phage display system was used for the random screening of peptides that facilitate EC uptake in conjunction with a pool of oligonucleotides randomly coding for 7-mer peptides. For amplification, the library was inoculated with BL21 culture (OD600 of 0.5–1.0) and induced with 1 mM IPTG at 37°C for 2 hours until cell lysiswas observed. The lysate containing phages was clarified by centrifugation at 8000 g for 10 minutes, the supernatant was titered, and aliquots were stored a 4°C. 527 AYPGX6 phage display library 10 2.0e8 Jing Yang (Research and Development, Bristol Myers Squibb Company, Princeton, USA) Linear The biased library was constructed by a single-stranded library oligonucleotide extension method. The sequence of the library oligonucleotide was 5’-CGGCCGAACC TCCACC(MNN)6A CCTGGATAAG CAGAGTGAGA ATAGAAAGGT ACC-3’ and the vector was a derivative of M13KE (New England Biolabs, Ipswich, MA) where the insert encoded GlyGly. 528 XCXmCXnCXoCX phage library pool 9-14 Harm-Anton Klok (Laboratoire des Polymères, Institut des Matériaux and Institut des Sciences et Ingénierie Chimiques, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland) Circular Peptide libraries XD9 to XD14 were constructed. The libraries had the peptide format of XCXmCXnCXoCX (m + n + o = 3, 4, 5, 6, 7 or 8; where X indicates random amino acids, C indicates cysteine). The libraries are constructed from a filamentous phage fd, which displays peptides on the N terminus of the pIII coat protein at the end of the viral particle. In total, five peptides are displayed per viral particle. Escherichia coli TG1: All phage display experiments utilized E. coli TG1 cells for phage amplification and titer.