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.