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.