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