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Identification of Novel Non-phosphorylated Ligands, Which Bind Selectively to the SH2 Domain of Grb7

2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês

10.1074/jbc.m111816200

ISSN

1083-351X

Autores

Stephanie C. Pero, Lyn Oligino, Roger J. Daly, Amy L. Soden, Chen Liu, Peter P. Roller, Peng Li, David N. Krag,

Tópico(s)

HER2/EGFR in Cancer Research

Resumo

Grb7 is an adapter-type signaling protein, which is recruited via its SH2 domain to a variety of receptor tyrosine kinases (RTKs), including ErbB2 and ErbB3. It is overexpressed in breast, esophageal, and gastric cancers, and may contribute to the invasive potential of cancer cells. Molecular interactions involving Grb7 therefore provide attractive targets for therapeutic intervention. We have utilized phage display random peptide libraries as a source of small peptide ligands to the SH2 domain of Grb7. Screening these libraries against purified Grb7 SH2 resulted in the identification of Grb7-binding peptide phage clones that contained a non-phosphorylated Tyr-X-Asn (Y XN) motif. The tyrosine-phosphorylated form of this motif is characteristic of Grb7 SH2 domain binding sites identified in RTKs and other signaling proteins such as Shc. Peptides that are non-phosphorylated have greater potential in the development of therapeutics because of the instability of a phosphate group in vivo. Using a biased library approach with this conserved Y XN motif, we identified seven different peptide phage clones, which bind specifically to the SH2 domain of Grb7. These peptides did not bind to the SH2 domain of Grb2 (which also selects for Asn at pY+2) or Grb14, a closely related family member. The cyclic structure of the peptides was required to bind to the Grb7 SH2 domain. Importantly, the synthetic Grb7-binding peptide G7-18 in cell lysates was able to specifically inhibit the association of Grb7 with the ErbB family of RTKs, in particular ErbB3, in a dose-dependent manner. These peptides will be useful in the development of targeted molecular therapeutics for cancers overexpressing Grb7 and in the development of Grb7-specific inhibitors to gain a complete understanding of the physiological role of Grb7. Grb7 is an adapter-type signaling protein, which is recruited via its SH2 domain to a variety of receptor tyrosine kinases (RTKs), including ErbB2 and ErbB3. It is overexpressed in breast, esophageal, and gastric cancers, and may contribute to the invasive potential of cancer cells. Molecular interactions involving Grb7 therefore provide attractive targets for therapeutic intervention. We have utilized phage display random peptide libraries as a source of small peptide ligands to the SH2 domain of Grb7. Screening these libraries against purified Grb7 SH2 resulted in the identification of Grb7-binding peptide phage clones that contained a non-phosphorylated Tyr-X-Asn (Y XN) motif. The tyrosine-phosphorylated form of this motif is characteristic of Grb7 SH2 domain binding sites identified in RTKs and other signaling proteins such as Shc. Peptides that are non-phosphorylated have greater potential in the development of therapeutics because of the instability of a phosphate group in vivo. Using a biased library approach with this conserved Y XN motif, we identified seven different peptide phage clones, which bind specifically to the SH2 domain of Grb7. These peptides did not bind to the SH2 domain of Grb2 (which also selects for Asn at pY+2) or Grb14, a closely related family member. The cyclic structure of the peptides was required to bind to the Grb7 SH2 domain. Importantly, the synthetic Grb7-binding peptide G7-18 in cell lysates was able to specifically inhibit the association of Grb7 with the ErbB family of RTKs, in particular ErbB3, in a dose-dependent manner. These peptides will be useful in the development of targeted molecular therapeutics for cancers overexpressing Grb7 and in the development of Grb7-specific inhibitors to gain a complete understanding of the physiological role of Grb7. SH2 domains are found in a large number of proteins and mediate signal transduction pathways associated with tyrosine kinase activity by binding to specific phosphorylated tyrosine residues on target proteins. These domains, ∼100 amino acids long, serve a critical role by transmitting signals within a cell, act as adapters between receptors and downstream signaling molecules, and regulate the kinase activity of specific proteins (1.Pawson T. Adv. Cancer Res. 1994; 64: 87-110Crossref PubMed Google Scholar, 2.Margolis B. Prog. Biophys. Mol. Biol. 1994; 62: 223-244Crossref PubMed Scopus (48) Google Scholar). These domains are attractive targets for developing targeted therapeutics, especially in the area of oncology and immunology. Our laboratory was the first to identify a non-phosphorylated ligand to the SH2 domain of Grb2 (3.Oligino L. Lung F.D. Sastry L. Bigelow J. Cao T. Curran M. Burke Jr., T.R. Wang S. Krag D. Roller P.P. King C.R. J. Biol. Chem. 1997; 272: 29046-29052Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), and several other groups have also reported ligands targeting the SH2 domains of Grb2 (4.Furet P. Gay B. Caravatti G. Garcia-Echeverria C. Rahuel J. Schoepfer J. Fretz H. J. Med. Chem. 1998; 41: 3442-3449Crossref PubMed Scopus (86) Google Scholar, 5.Hart C.P. Martin J.E. Reed M.A. Keval A.A. Pustelnik M.J. Northrop J.P. Patel D.V. Grove J.R. Cell. Signal. 1999; 11: 453-464Crossref PubMed Scopus (25) Google Scholar), pp60c-src (6.Buchanan J.L. Bohacek R.S. Luke G.P. Hatada M. Lu X. Dalgarno D.C. Narula S.S. Yuan R. Holt D.A. Bioorg. Med. Chem. Lett. 1999; 9: 2353-2358Crossref PubMed Scopus (42) Google Scholar, 7.Gilmer T. Rodriguez M. Jordan S. Crosby R. Alligood K. Green M. Kimery M. Wagner C. Kinder D. Charifson P. J. Biol. Chem. 1994; 269: 31711-31719Abstract Full Text PDF PubMed Google Scholar, 8.Pacofsky G.J. Lackey K. Alligood K.J. Berman J. Charifson P.S. Crosby R.M. 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Chem. 1999; 42: 1757-1766Crossref PubMed Scopus (69) Google Scholar). Our interests are to target SH2 domain proteins that play a role in signaling by the ErbB tyrosine kinase family of receptors, which comprises EGFR, ErbB2, ErbB3, and ErbB4. Grb7 is an especially promising cancer target because the encoding gene maps closely to the ErbB2 gene and is found co-amplified in a subset of breast cancers (12.Stein D. Wu J. Fuqua S.A. Roonprapunt C. Yajnik V. D'Eustachio P. Moskow J.J. Buchberg A.M. Osborne C.K. Margolis B. EMBO J. 1994; 13: 1331-1340Crossref PubMed Scopus (220) Google Scholar). In addition, Grb7 is overexpressed with ErbB2 in breast, esophageal, and gastric cancers (12.Stein D. Wu J. Fuqua S.A. Roonprapunt C. Yajnik V. D'Eustachio P. Moskow J.J. Buchberg A.M. Osborne C.K. Margolis B. EMBO J. 1994; 13: 1331-1340Crossref PubMed Scopus (220) Google Scholar, 13.Kishi T. Sasaki H. Akiyama N. Ishizuka T. Sakamoto H. Aizawa S. Sugimura T. Terada M. Biochem. Biophys. Res. 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Blocking the function of ErbB2 inhibits proliferation of cancer cells as demonstrated in preclinical (18.Fendly B.M. Winget M. Hudziak R.M. Lipari M.T. Napier M.A. Ullrich A. Cancer Res. 1990; 50: 1550-1558PubMed Google Scholar, 19.Hudziak R.M. Lewis G.D. Winget M. Fendly B.M. Shepard H.M. Ullrich A. Mol. Cell. Biol. 1989; 9: 1165-1172Crossref PubMed Scopus (664) Google Scholar) and clinical studies (20.Slamon D.J. Leyland-Jones B. Shak S. Fuchs H. Paton V. Bajamonde A. Fleming T. Eiermann W. Wolter J. Pegram M. Baselga J. Norton L. N. Engl. J. Med. 2001; 344: 783-792Crossref PubMed Scopus (9491) Google Scholar, 21.Baselga J. Tripathy D. Mendelsohn J. Baughman S. Benz C.C. Dantis L. Sklarin N.T. Seidman A.D. Hudis C.A. Moore J. Rosen P.P. Twaddell T. Henderson I.C. Norton L. J. Clin. Oncol. 1996; 14: 737-744Crossref PubMed Scopus (1273) Google Scholar). In addition, co-expression of Grb7 with ErbB2 was detected in 31% of esophageal carcinomas and was significantly correlated with extramucosal tumor invasion (14.Tanaka S. Mori M. Akiyoshi T. Tanaka Y. Mafune K. Wands J.R. Sugimachi K. Cancer Res. 1997; 57: 28-31PubMed Google Scholar). Moreover, Grb7 is co-expressed with ErbB3 and ErbB4, which are known to heterodimerize with ErbB2, in a subgroup of human breast cancer cell lines (22.Fiddes R.J. Campbell D.H. Janes P.W. Sivertsen S.P. Sasaki H. Wallasch C. Daly R.J. J. Biol. Chem. 1998; 273: 7717-7724Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The SH2 domain of Grb7 binds to an intracellular phosphorylated tyrosine residue of ErbB2 as well as to specific phosphotyrosines of other receptors such as ErbB3 (22.Fiddes R.J. Campbell D.H. Janes P.W. Sivertsen S.P. Sasaki H. Wallasch C. Daly R.J. J. Biol. Chem. 1998; 273: 7717-7724Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), ErbB4 (22.Fiddes R.J. Campbell D.H. Janes P.W. Sivertsen S.P. Sasaki H. Wallasch C. Daly R.J. J. Biol. Chem. 1998; 273: 7717-7724Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), platelet-derived growth factor receptor (23.Yokote K. Margolis B. Heldin C.H. Claesson-Welsh L. J. Biol. Chem. 1996; 271: 30942-30949Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), focal adhesion kinase (24.Han D.C. Guan J.L. J. Biol. Chem. 1999; 274: 24425-24430Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), c-Kit stem cell factor receptor (25.Thommes K. Lennartsson J. Carlberg M. Ronnstrand L. Biochem. J. 1999; 341: 211-216Crossref PubMed Scopus (101) Google Scholar, 26.Pandey A. Liu X. Dixon J.E. Di Fiore P.P. Dixit V.M. J. Biol. Chem. 1996; 271: 10607-10610Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), Ret (26.Pandey A. Liu X. Dixon J.E. Di Fiore P.P. Dixit V.M. J. Biol. Chem. 1996; 271: 10607-10610Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and Tek/Tie (27.Jones N. Master Z. Jones J. Bouchard D. Gunji Y. Sasaki H. Daly R. Alitalo K. Dumont D.J. J. Biol. Chem. 1999; 274: 30896-30905Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The Grb7 SH2 domain also binds to the protein tyrosine phosphatase SHPTP2 (28.Keegan K. Cooper J.A. Oncogene. 1996; 12: 1537-1544PubMed Google Scholar) and the adapter protein SHC (12.Stein D. Wu J. Fuqua S.A. Roonprapunt C. Yajnik V. D'Eustachio P. Moskow J.J. Buchberg A.M. Osborne C.K. Margolis B. EMBO J. 1994; 13: 1331-1340Crossref PubMed Scopus (220) Google Scholar). The functional consequences of the association of Grb7 with its physiological ligands have not been completely elucidated. However, the importance of Grb7 in tumor progression has been suggested by several studies (14.Tanaka S. Mori M. Akiyoshi T. Tanaka Y. Mafune K. Wands J.R. Sugimachi K. Cancer Res. 1997; 57: 28-31PubMed Google Scholar, 24.Han D.C. Guan J.L. J. Biol. Chem. 1999; 274: 24425-24430Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 29.Tanaka S. Mori M. Akiyoshi T. Tanaka Y. Mafune K. Wands J.R. Sugimachi K. J. Clin. Invest. 1998; 102: 821-827Crossref PubMed Scopus (67) Google Scholar, 30.Tanaka S. Sugimachi K. Kawaguchi H. Saeki H. Ohno S. Wands J.R. J. Cell. Physiol. 2000; 183: 411-415Crossref PubMed Scopus (51) Google Scholar). In one recent study, histologic expression of Grb7 in primary squamous cell esophageal tumors at the time of surgical resection showed Grb7 to be overexpressed in 14 of 31 esophageal carcinomas as compared with adjacent normal mucosa. The overexpression of Grb7 in these histological sections was significantly correlated with the presence of lymph node metastases (30.Tanaka S. Sugimachi K. Kawaguchi H. Saeki H. Ohno S. Wands J.R. J. Cell. Physiol. 2000; 183: 411-415Crossref PubMed Scopus (51) Google Scholar). In another study, inducible Grb7 overexpression in NIH3T3 cells increased cell migration toward fibronectin, whereas overexpression of its SH2 domain inhibited cell migration (24.Han D.C. Guan J.L. J. Biol. Chem. 1999; 274: 24425-24430Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Likewise, overexpression of Grb7, and not a Grb7 mutant where the SH2 and GM domains were eliminated, stimulated cell migration in CHO cells (31.Han D.C. Shen T.L. Guan J.L. J. Biol. Chem. 2000; 275: 28911-28917Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Furthermore, in an esophageal cancer line, down-regulation of Grb7 using antisense technology inhibited the invasive properties of the cells (29.Tanaka S. Mori M. Akiyoshi T. Tanaka Y. Mafune K. Wands J.R. Sugimachi K. J. Clin. Invest. 1998; 102: 821-827Crossref PubMed Scopus (67) Google Scholar). Another promising feature of Grb7 as a tumor target is its limited tissue distribution, which is unlike that of many other SH2 domain-containing proteins which are ubiquitously expressed (2.Margolis B. Prog. Biophys. Mol. Biol. 1994; 62: 223-244Crossref PubMed Scopus (48) Google Scholar). Having limited tissue distribution is advantageous for targeting therapeutics to cancer cells. The fact that Grb7 is associated with tumor-related molecules and has been demonstrated to have a direct role in cancer cell migration makes it a potentially important therapeutic target. Phage display libraries have been used extensively to identify peptide ligands for a variety of molecular targets (32.Zwick M.B. Shen J. Scott J.K. Curr. Opin. Biotechnol. 1998; 9: 427-436Crossref PubMed Scopus (117) Google Scholar, 33.Scott J.K. Craig L. Curr. Opin. Biotechnol. 1994; 5: 40-48Crossref PubMed Scopus (87) Google Scholar, 34.Nilsson F. Tarli L. Viti F. Neri D. Adv. 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Science. 1998; 279: 377-380Crossref PubMed Scopus (1873) Google Scholar); 2) specifically bind and regulate estrogen receptor α transcriptional activity, which can be used in the development of novel treatments for tamoxifen-refractory breast cancers (38.Norris J.D. Paige L.A. Christensen D.J. Chang C.Y. Huacani M.R. Fan D. Hamilton P.T. Fowlkes D.M. McDonnell D.P. Science. 1999; 285: 744-746Crossref PubMed Scopus (335) Google Scholar); and 3) specifically bind to type IV collagenase/gelatinase matrix metalloproteinases and inhibit the migration of human endothelial cells and tumor cells, which may prove useful in tumor targeting and anti-cancer therapies (39.Koivunen E. Arap W. Valtanen H. Rainisalo A. Medina O.P. Heikkila P. Kantor C. Gahmberg C.G. Salo T. Konttinen Y.T. Sorsa T. Ruoslahti E. Pasqualini R. Nat. Biotechnol. 1999; 17: 768-774Crossref PubMed Scopus (516) Google Scholar). Peptides identified from phage display libraries can be modified, if necessary, to obtain an intracellularly stable, cell permeable, and higher affinity peptide that maintains specific binding to its target. These modifications can be achieved utilizing a homologous-analog and/or peptidomimetics approach to confer desirable pharmaceutical properties of stability, affinity, and specificity (40.Burke Jr., T.R. Smyth M.S. Otaka A. Nomizu M. Roller P.P. Wolf G. Case R. Shoelson S.E. Biochemistry. 1994; 33: 6490-6494Crossref PubMed Scopus (199) Google Scholar, 41.Ye B. Akamatsu M. Shoelson S.E. Wolf G. Giorgetti-Peraldi S. Yan X. Roller P.P. Burke Jr., T.R. J. Med. Chem. 1995; 38: 4270-4275Crossref PubMed Scopus (74) Google Scholar, 42.Yao Z.J. King C.R. Cao T. Kelley J. Milne G.W. Voigt J.H. Burke Jr., T.R. J. Med. Chem. 1999; 42: 25-35Crossref PubMed Scopus (94) Google Scholar, 43.Garcia-Echeverria C. Furet P. Gay B. Fretz H. Rahuel J. Schoepfer J. Caravatti G. J. Med. 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In addition, a structure-based approach can be used for optimization of the targeting properties of a peptide, such as affinity, by studying the peptide or the peptide complexed to its target (4.Furet P. Gay B. Caravatti G. Garcia-Echeverria C. Rahuel J. Schoepfer J. Fretz H. J. Med. Chem. 1998; 41: 3442-3449Crossref PubMed Scopus (86) Google Scholar, 6.Buchanan J.L. Bohacek R.S. Luke G.P. Hatada M. Lu X. Dalgarno D.C. Narula S.S. Yuan R. Holt D.A. Bioorg. Med. Chem. Lett. 1999; 9: 2353-2358Crossref PubMed Scopus (42) Google Scholar, 49.Schoepfer J. Gay B. Caravatti G. Garcia-Echeverria C. Fretz H. Rahuel J. Furet P. Bioorg. Med. Chem. Lett. 1998; 8: 2865-2870Crossref PubMed Scopus (25) Google Scholar, 50.Furet P. Garcia-Echeverria C. Gay B. Schoepfer J. Zeller M. Rahuel J. J. Med. Chem. 1999; 42: 2358-2363Crossref PubMed Scopus (56) Google Scholar). Using phage display, we have identified the first non-phosphorylated peptides that are highly selective for the Grb7 SH2 domain compared with Grb2 or Grb14 SH2 domains. In addition, one of the Grb7-binding peptides is able to effectively block Grb7 from binding to the ErbB family of RTKs. 1The abbreviations used are: RTKsreceptor tyrosine kinasesGSTglutathione S-transferaseELISAenzyme-linked immunosorbent assayFmocN-(9 fluorenyl)methoxycarbonylpYphosphotyrosineNAno armsSOC2% (w/v) Bacto-tryptone, 0.5% (w/v) Bacto-yeast extract, 10 mm NaCl, 2.5 mm KCl, 10 mm MgCl, 10 mm MgSO4, and 20 mm glucoseNNKa cloning scheme in which N represents equal amounts of G, A, T, or C and K represents equal amounts of G or T These Grb7-binding peptides will be further evaluated as lead compounds for drugs that target diseases where Grb7 is overexpressed and may provide useful tools for the elucidation of Grb7 function. receptor tyrosine kinases glutathione S-transferase enzyme-linked immunosorbent assay N-(9 fluorenyl)methoxycarbonyl phosphotyrosine no arms 2% (w/v) Bacto-tryptone, 0.5% (w/v) Bacto-yeast extract, 10 mm NaCl, 2.5 mm KCl, 10 mm MgCl, 10 mm MgSO4, and 20 mm glucose a cloning scheme in which N represents equal amounts of G, A, T, or C and K represents equal amounts of G or T The peptide libraries used in these studies were constructed in the fUSE5 gene III phage display system (51.Scott J.K. Smith G.P. Science. 1990; 249: 386-390Crossref PubMed Scopus (1900) Google Scholar). The fUSE5 vector and Escherichia coli host strains (K91 Kan and MC1061F′) were the generous gift of Dr. George Smith at the University of Missouri (51.Scott J.K. Smith G.P. Science. 1990; 249: 386-390Crossref PubMed Scopus (1900) Google Scholar). The half-site cloning method used by Cwirla et al. (52.Cwirla S.E. Peters E.A. Barrett R.W. Dower W.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6378-6382Crossref PubMed Scopus (854) Google Scholar) was employed in construction of these libraries. The two half-site oligos 5′-TCCGCCAGCCCCGT-3′ (GS5′) and 5′-CGGCCCCGCCTCC-3′(GS3′) are complementary to the 5′- and 3′-ends of the peptide insert-encoding oligonucleotide. The random peptide encoding oligonucleotide has the following sequence: 5′pGGGCTGGCGGA(NNK)4TGC(NNK)10TGC(NNK)4GGAGGCGGGGCCGCTG 3′ (referred to as ON-10). The Y XN-biased peptide encoding oligonucleotide has the following sequence: GGGCTGGCGGA(NNK)4TGC(NNK)4TAYEFJAAY(NNK)3TGC(NNK)4GGAGGCGGGGCCGCTG (referred to as ON-Y XN) where N refers to equal amounts of G, A, T or C; K refers to equal amounts of G or T; Y refers to equal amounts of T and C; E refers to 62.5% G, 12.5% A, 12.5% C, and 12.5% T; F refers to 37.5% A, 37.5% C, 12.5% G, and 12.5% T; and J refers to 37.5% G, 37.5% T, 12.5% A, and 12.5% C. Oligos GS5′, GS3′, and ON-10 were synthesized by the University of Vermont Molecular Genetics Oligo Synthesis Facility, and the Y XN-biased oligonucleotide by Biosynthesis, Inc. (Lewisville, Texas). The annealing reaction for the random library was carried out by mixing 219 pmol of GS5′, 219pmol GS3′, and 10.96 pmol of ON-10. The annealing reaction for the Y XN-biased library contained 144 pmol of GS5′, 144 pmol of GS3′, and 7.5 pmol of ON-YXN. After the annealing reactions were carried out to generate double-stranded termini, the non-annealed single stranded half-site oligos were removed using QIAquick Clean-up Kit (Qiagen), eluting in 36 μl of 10 mmTris-HCl buffer, pH 8.5. The SfiI-digested fUSE5 phage vector (13.7 μg for the random peptide library and 15 μg for the Y XN-biased library) were ligated to the annealed oligos in a 1-ml reaction containing T4 DNA Ligase (Invitrogen) (1 unit/μg of vector) overnight at 15 °C followed by heat inactivation at 65 °C for 10 min. The ligation reactions were ethanol-precipitated with pellet paint co-precipitant (Novagen) for 3 h at −20 °C, resuspended in 60 μl of water, and heated at 50 °C for 5 min to thoroughly dissolve the DNA. The ligations were transformed into freshly prepared electrocompetent E. coli (MC1061F′) cells. Sixty electrotransformations, each containing 40 μl of cells, were performed at 1.7 kV. After transformation, the cells were collected in 2 ml of SOC medium and grown for an hour at 37 °C, 220 rpm. After the hour of non-selective growth, all transformation reactions were pooled, centrifuged at 8000 rpm for 10 min, and resuspended in 10 ml of SOC medium. An aliquot of the transformations was plated onto an LB/tetracycline plate to determine the original number of transformants. The pools of transformants were plated on Pyrex dishes (11 × 7 inches) containing 2× YT/tetracycline/ streptomycin (1 ml per dish). The dishes were incubated at 37 °C overnight to allow and produce thousands of copies of each peptide phage particle library member. The SH2 domain constructs for Grb7, Grb14, Grb7 βD5/βD6, and Grb2 were provided by Dr. Roger Daly. The construction of these plasmids have been described by Janes et al. (53.Janes P.W. Lackmann M. Church W.B. Sanderson G.M. Sutherland R.L. Daly R.J. J. Biol. Chem. 1997; 272: 8490-8497Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and Lowenstein et al. (54.Lowenstein E.J. Daly R.J. Batzer A.G. Li W. Margolis B. Lammers R. Ullrich A. Skolnik E.Y. Bar-Sagi D. Schlessinger J. Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1348) Google Scholar). These proteins were expressed in a GST fusion bacterial expression system using the pGEX-2T expression vector (Amersham Biosciences, Inc.) and purified from isopropyl-β-d-thiogalactopyranoside-induced bacterial cultures as described previously (55.Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). Purity was determined by SDS-PAGE. Protein concentration was determined by Bradford-based protein assay (Bio-Rad). The libraries were amplified so that there were ∼1000 transforming units of each peptide phage representing 200 library equivalents. The amplified phage were infected into K91/Kan cells, plated onto five 2× YT agar dishes (11 × 7 inches) containing 40 μg/ml tetracycline, incubated overnight at 30 °C, and harvested in 1× phosphate-buffered saline with a prokaryotic protease inhibitor mixture (1:1000)(Sigma) by sweeping the bacteria off the agar with a bent glass rod. The bacterial cell suspension was centrifuged at 8000 rpm for 10 min at 4 °C, and the supernatant was filtered through a 0.22-μm polyethersulfone (PES) filter. The filtrate was incubated for 30 min on ice with 0.15 volume of polyethylene glycol/NaCl to precipitate the phage and centrifuged at 9000 rpm, 20 min, 4 °C. The phage pellet was resuspended in 1× phosphate-buffered saline with a prokaryotic protease inhibitor mixture. GST-binding peptide phage were subtracted from the library by preincubation of the library with 2 μg of GST immobilized on 20 μl of glutathione-Sepharose (Amersham Biosciences, Inc.). The phage were screened by adding them to 12 μg of purified GST·Grb7 SH2 immobilized onto 20 μl of glutathione-Sepharose and incubated for 2 h on a nutator at 25 °C. The unbound phage were removed by washing five times with 1× phosphate-buffered saline with a prokaryotic protease inhibitor mixture. Phage binders were eluted twice with a low pH (2.3) buffer followed by two elutions with a high pH (12.Stein D. Wu J. Fuqua S.A. Roonprapunt C. Yajnik V. D'Eustachio P. Moskow J.J. Buchberg A.M. Osborne C.K. Margolis B. EMBO J. 1994; 13: 1331-1340Crossref PubMed Scopus (220) Google Scholar) buffer. K91/Kan cells were then infected with the pooled phage elutions, and a small aliquot was plated to determine the total amount of phage particles bound to the targets. The remaining phage were amplified overnight, harvested, screened, and eluted as described above. To determine the amount of enrichment after the second and third rounds of panning, half of the pre-cleared phage were added to fresh GST immobilized on glutathione beads to determine the amount of phage binding GST-glutathione-Sepharose compared with GST·Grb7 SH2 glutathione-Sepharose. Three rounds of screening were carried out before clones were sequenced or tested in an ELISA. Isolated peptide phage-infected E. coli colonies were grown in 5 ml of LB/tetracycline broth. The double-stranded phage replicative form DNA was isolated using the QIAprep Spin Miniprep kit (Qiagen). The following sequencing primer designed by George Smith was used to sequence the random insert in the fUSE5 vector: 5′-CCC TCA TAG TTA GCG TAA CG-3′ (51.Scott J.K. Smith G.P. Science. 1990; 249: 386-390Crossref PubMed Scopus (1900) Google Scholar). The sequence reactions were carried out using BigDye Version 1 Dye Terminator kit (PerkinElmer Life Sciences). The sequencing reactions and automated DNA sequencing were performed in the Vermont Cancer Center DNA Analysis Facility. The amino acid sequences of peptides displayed by peptide phage were deduced from the DNA sequence of the corresponding phage clones. Consensus sequence identification was performed by visual inspection and with the ClustalW alignment program. A reverse phage ELISA was used to evaluate the ability of individual phage clones to bind to Grb7 SH2 (32.Zwick M.B. Shen J. Scott J.K. Curr. Opin. Biotechnol. 1998; 9: 427-436Crossref PubMed Scopus (117) Google Scholar, 56.Valadon P. Scharff M.D. J. Immunol. Methods. 1996; 197: 171-179Crossref PubMed Scopus (28) Google Scholar). Briefly, protein targets (5 μg/ml) were added to a 96-well Maxisorp plate (Nunc) plate precoated with anti-GST polyclonal antibody (1 μg/ml final concentration) (Amersham Biosciences, Inc.) and blocked with casein in Tris-buffered saline (Pierce). The peptide phage were concentrated with polyethylene glycol/NaCl precipitation methods (51.Scott J.K. Smith G.P. Science. 1990; 249: 386-390Crossref PubMed Scopus (1900) Google Scholar), added to each well (1 × 107−1 × 108 phage/well), and incubated for 2 h. at room temperature. Unbound phage were removed with 0.1% Tween-Tris-buffered saline buffer, and phage were detected with horseradish peroxidase-conjugated anti-M13 monoclonal antibody (1:1000) (Amersham Biosciences, Inc.) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate (Calbiochem). Plates were read at A405. To determine whether the four

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