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• W2041504987 abstract "Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, is unique in its ability and mechanism of inhibiting serine proteases of a broad range of substrate specificity. However, although the catalytic domain of human urokinase-type plasminogen activator (uPA) has 40% identity to bovine trypsin and the substrate specificities of these two proteases are virtually identical, ecotin inhibits uPA almost 10,000-fold less efficiently than trypsin. Ecotin was expressed on the surface of filamentous bacteriophage (ecotin phage) to allow the isolation of more potent inhibitors of uPA from a library of ecotin variants. The 142-amino acid inhibitor was fused to the C-terminal domain of the M13 minor coat protein, pIII, through a Gly-Gly-Gly linker and assembled into phage particles. The ecotin phage were shown to react with anti-ecotin antibodies, revealing a stoichiometry of approximately one ecotin per bacteriophage. The ecotin displayed on the surface of phage inhibited trypsin with an equilibrium dissociation constant of 6.7 nM, in close approximation to that of free ecotin, indicating that phage-associated ecotin is correctly folded and functionally active. Reactive-site amino acids 84 and 85 of ecotin were then randomized and a library of 400 unique ecotin phage was created. Three hundred thousand members of the library were screened with immobilized uPA and subjected to three rounds of binding and in vitro selection. DNA sequence analysis of the selected ecotin phage showed that ecotin M84R/M85R predominated while ecotin M84R, M84K, and M84R/M85K were present at a lower frequency. The four ecotin variants were overexpressed and purified and their affinities toward uPA were determined. Each of the selected ecotin variants exhibited increased affinity for uPA when compared to wild-type ecotin with ecotin M84R/M85R showing a 2800-fold increase in binding affinity. Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, is unique in its ability and mechanism of inhibiting serine proteases of a broad range of substrate specificity. However, although the catalytic domain of human urokinase-type plasminogen activator (uPA) has 40% identity to bovine trypsin and the substrate specificities of these two proteases are virtually identical, ecotin inhibits uPA almost 10,000-fold less efficiently than trypsin. Ecotin was expressed on the surface of filamentous bacteriophage (ecotin phage) to allow the isolation of more potent inhibitors of uPA from a library of ecotin variants. The 142-amino acid inhibitor was fused to the C-terminal domain of the M13 minor coat protein, pIII, through a Gly-Gly-Gly linker and assembled into phage particles. The ecotin phage were shown to react with anti-ecotin antibodies, revealing a stoichiometry of approximately one ecotin per bacteriophage. The ecotin displayed on the surface of phage inhibited trypsin with an equilibrium dissociation constant of 6.7 nM, in close approximation to that of free ecotin, indicating that phage-associated ecotin is correctly folded and functionally active. Reactive-site amino acids 84 and 85 of ecotin were then randomized and a library of 400 unique ecotin phage was created. Three hundred thousand members of the library were screened with immobilized uPA and subjected to three rounds of binding and in vitro selection. DNA sequence analysis of the selected ecotin phage showed that ecotin M84R/M85R predominated while ecotin M84R, M84K, and M84R/M85K were present at a lower frequency. The four ecotin variants were overexpressed and purified and their affinities toward uPA were determined. Each of the selected ecotin variants exhibited increased affinity for uPA when compared to wild-type ecotin with ecotin M84R/M85R showing a 2800-fold increase in binding affinity. Urokinase-type plasminogen activator (uPA)1 1The abbreviations used here are: uPAurokinase-type plasminogen activatorcfuampicillin-resistant colony forming unitEGFepidermal growth factorIPTGisopropylthio-β-D-galactosideLMuPAlow molecular weight urokinase-type plasminogen activatorpIIIgene III product of M13 bacteriophagePAI-1type 1 plasminogen activator inhibitortPAtissue-type plasminogen activatorZcarbobenzoxy. and tissue-type plasminogen activator (tPA) are two serine proteases that catalyze the conversion of the inactive precursor plasminogen, to plasmin, a serine protease of broad substrate specificity (1Haber E. Quertermous T. Matsueda G.R. Runge M.S. Science. 1989; 243: 51-56Crossref PubMed Scopus (124) Google Scholar, 2Mayer M. Clin. Biochem. 1990; 23: 197-211Crossref PubMed Scopus (125) Google Scholar). uPA has been found to be involved in the activation of pericellular proteolysis during cell migration and tissue remodeling, while the function of tPA is primarily connected to intravascular clot dissolution (3Dano K. Andreasen P.A. Grondahl-Hansen J. Kristensen P. Nielsen L.S. Skriver L. Adv. Cancer Res. 1985; 44: 139-266Crossref PubMed Scopus (2298) Google Scholar, 4Saksela O. Rifkin D.B. Annu. Rev. Cell Biol. 1988; 4: 93-126Crossref PubMed Scopus (717) Google Scholar). Although no three-dimensional structure currently exists for human uPA, it is thought to be composed of three domains: an NH2-terminal domain (residues 1-45), which has partial homology to EGF (5Gunzler W.A. Steffens G.J. Otting F. Kim S.M. Frankus E. Flohe L. Hoppe-Seyler's Z Physiol. Chem. 1982; 363: 1155-1165Crossref PubMed Scopus (259) Google Scholar, 6Appella E. Robinson E.A. Ullrich S.J. Stoppelli M.P. Corti A. Cassani G. Blasi F. J. Biol. Chem. 1987; 262: 4437-4440Abstract Full Text PDF PubMed Google Scholar) followed by a kringle domain (residues 50-131) (5Gunzler W.A. Steffens G.J. Otting F. Kim S.M. Frankus E. Flohe L. Hoppe-Seyler's Z Physiol. Chem. 1982; 363: 1155-1165Crossref PubMed Scopus (259) Google Scholar), and a COOH-terminal protease domain (residues 159-411) with 40% identity to trypsin (7Steffens G.J. Gunzler W.A. Otting F. Frankus E. Flohe L. Hoppe-Seyler's Z Physiol. Chem. 1982; 363: 1043-1058Crossref PubMed Scopus (174) Google Scholar). The EGF-like domain of uPA binds the membrane-bound urokinase receptor (8Vassalli J.D. Baccino D. Belin D. J. Cell Biol. 1985; 100: 86-92Crossref PubMed Scopus (589) Google Scholar), localizing the proteolytic activity of uPA to the cell surface. Receptor-bound uPA has been shown to cleave a 66-kDa extracellular matrix protein (9Keski-Oja J. Vaheri A. Biochim. Biophys. Acta. 1982; 720: 141-146Crossref PubMed Scopus (48) Google Scholar) as well as fibronectin (10Quigley J.P. Gold L.I. Schwimmer R. Sullivan L.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2776-2780Crossref PubMed Scopus (129) Google Scholar). The presence of uPA on the cell surface also allows the formation of cell-surface plasmin, which is capable of degrading most components of the extracellular matrix, either directly or through activation of procollagenases (3Dano K. Andreasen P.A. Grondahl-Hansen J. Kristensen P. Nielsen L.S. Skriver L. Adv. Cancer Res. 1985; 44: 139-266Crossref PubMed Scopus (2298) Google Scholar). Cell surface uPA has been implicated in mediating processes such as tumor growth, cell invasion, metastasis, cell migration, and tissue remodeling (3Dano K. Andreasen P.A. Grondahl-Hansen J. Kristensen P. Nielsen L.S. Skriver L. Adv. Cancer Res. 1985; 44: 139-266Crossref PubMed Scopus (2298) Google Scholar, 11Testa J.E. Quigley J.P. Cancer Metastasis Rev. 1990; 9: 353-367Crossref PubMed Scopus (152) Google Scholar), all of which require extracellular proteolytic activity. urokinase-type plasminogen activator ampicillin-resistant colony forming unit epidermal growth factor isopropylthio-β-D-galactoside low molecular weight urokinase-type plasminogen activator gene III product of M13 bacteriophage type 1 plasminogen activator inhibitor tissue-type plasminogen activator carbobenzoxy. High levels of receptor-bound uPA are found on the surface of many cancer cells (12Kirchheimer J.C. Wojta J. Christ G. Binder B.R. FASEB J. 1987; 1: 125-128Crossref PubMed Scopus (134) Google Scholar, 13Kirchheimer J.C. Nong Y.H. Remold H.G. J. Immunol. 1988; 141: 4229-4234PubMed Google Scholar, 14Nielsen L.S. Kellerman G.M. Behrendt N. Picone R. Dano K. Blasi F. J. Biol. Chem. 1988; 263: 2358-2363Abstract Full Text PDF PubMed Google Scholar). The role of uPA and its receptor in tumor invasion and metastasis suggests two possible approaches for chemotherapeutic intervention: one by blocking specific interactions between the EGF-like domain of uPA and the uPA receptor, and the other by specific inhibition of the proteolytic activity of uPA. A truncated, soluble form of the uPA receptor was produced genetically and shown to reduce the amount of uPA that bound to cells expressing wild-type uPA receptor (15Masucci M.T. Pedersen N. Blasi F. J. Biol. Chem. 1991; 266: 8655-8658Abstract Full Text PDF PubMed Google Scholar). By acting as a scavenger for uPA, the soluble uPA receptor also inhibited the proliferation and invasion of human cancer cells (16Wilhelm O. Weidle U. Hohl S. Rettenberger P. Schmitt M. Graeff H. FEBS Lett. 1994; 337: 131-134Crossref PubMed Scopus (104) Google Scholar). Alternatively, a uPA mutant which lacked proteolytic activity while retaining full receptor binding affinity was shown to compete for cell surface receptors and, in turn, inhibit metastasis (17Crowley C.W. Cohen R.L. Lucas B.K. Liu G. Shuman M.A. Levinson A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5021-5025Crossref PubMed Scopus (367) Google Scholar). Finally, high-affinity urokinase receptor antagonists were identified from a pentadecamer random peptide library and were shown to compete with the EGF-like domain of uPA for binding to the uPA receptor (18Goodson R.J. Doyle M.V. Kaufman S.E. Rosenberg S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7129-7133Crossref PubMed Scopus (199) Google Scholar). Although a certain extent of success in anti-invasion and anti-metastasis has been achieved in vitro using this approach, rapid clearance of uPA, uPA receptor derivatives, or natural peptides may pose a problem if they are used as therapeutic agents. Less effort has been made toward developing specific inhibitors for uPA, presumably because of the difficulty of discriminating uPA from other serine proteases. Of the synthetic uPA inhibitors that have been described to date, the 4-substituted benzo[b]thiophene-2-carboxamidines were the most potent and were shown to inhibit cell surface uPA as well as cell surface uPA-mediated fibronectin degradation (19Towle M.J. Lee A. Maduakor E.C. Schwartz C.E. Bridges A.J. Littlefield B.A. Cancer Res. 1993; 53: 2553-2559PubMed Google Scholar). The natural macromolecular inhibitor of the plasminogen activators is the type 1 plasminogen activator inhibitor (PAI-1), a single-chain glycoprotein with a molecular mass of approximately 50 kDa (20van Mourik J.A. Lawrence D.A. Loskutoff D.J. J. Biol. Chem. 1984; 259: 14914-14921Abstract Full Text PDF PubMed Google Scholar). However, PAI-1 does not discriminate between the plasminogen activators, inactivating tPA and uPA with nearly identical secondary rate constants (21York J.D. Li P. Gardell S.J. J. Biol. Chem. 1991; 266: 8495-8500Abstract Full Text PDF PubMed Google Scholar). Furthermore, high PAI-1 levels have recently been found to associate with malignancy in a number of cancers (22Grondahl-Hansen J. Christensen I.J. Rosenquist C. Brunner N. Mouridsen H.T. Dano K. Blichert-Toft M. Cancer Res. 1993; 53: 2513-2521PubMed Google Scholar, 23Janicke F. Schmitt M. Pache L. Ulm K. Harbeck N. Hofler H. Graeff H. Breast Cancer Res. Treat. 1993; 24: 195-208Crossref PubMed Scopus (358) Google Scholar, 24Foekens J.A. Schmitt M. van Putten W.L. Peters H.A. Kramer M.D. Janicke F. Klijn J.G. J. Clin. Oncol. 1994; 12: 1648-1658Crossref PubMed Scopus (228) Google Scholar, 25Pedersen H. Grondahl-Hansen J. Francis D. Osterlind K. Hansen H.H. Dano K. Brunner N. Cancer Res. 1994; 54: 120-123PubMed Google Scholar). These findings suggest that, in addition to functioning as a uPA inhibitor in normal cells, PAI-1 or its complex may play a role in promoting growth or spreading of cancers. These attributes disfavor the use of PAI-1 as a therapeutic agent. Ecotin is a dimeric serine protease inhibitor found in the periplasm of Escherichia coli, where each unit of the dimer contains 142 amino acids (26Chung C.H. Ives H.E. Almeda S. Goldberg A.L. J. Biol. Chem. 1983; 258: 11032-11038Abstract Full Text PDF PubMed Google Scholar, 27McGrath M.E. Hines W.M. Sakanari J.A. Fletterick R.J. Craik C.S. J. Biol. Chem. 1991; 266: 6620-6625Abstract Full Text PDF PubMed Google Scholar). Ecotin has been found to inhibit pancreatic serine proteases of a broad range of specificity but not any known proteases from E. coli(26Chung C.H. Ives H.E. Almeda S. Goldberg A.L. J. Biol. Chem. 1983; 258: 11032-11038Abstract Full Text PDF PubMed Google Scholar). Recently, ecotin has also been found to be a highly potent anticoagulant and a reversible tight-binding inhibitor of human factor Xa (28Seymour J.L. Lindquist R.N. Dennis M.S. Moffat B. Yansura D. Reilly D. Wessinger M.E. Lazarus R.A. Biochemistry. 1994; 33: 3949-3958Crossref PubMed Scopus (71) Google Scholar). Ecotin belongs to the “substrate-like” class of inhibitors (29Laskowski Jr., M. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Crossref PubMed Scopus (1936) Google Scholar) with Met-84 at the reactive-site (the P1 site) (27McGrath M.E. Hines W.M. Sakanari J.A. Fletterick R.J. Craik C.S. J. Biol. Chem. 1991; 266: 6620-6625Abstract Full Text PDF PubMed Google Scholar). A crystal structure of ecotin complexed with trypsin showed that two trypsin molecules bind to an ecotin dimer in a 2-fold symmetry (30McGrath M.E. Erpel T. Bystroff C. Fletterick R.J. EMBO J. 1994; 13: 1502-1507Crossref PubMed Scopus (71) Google Scholar). In addition to the interactions through a primary site that includes the reactive-site loop, ecotin makes a total of 9 hydrogen bonds to trypsin through a secondary binding site located at the distal end of ecotin relative to the reactive site. Modeling studies with ecotin and other proteases including chymotrypsin and elastase indicates that similar interactions could occur, along with other unique contacts. The chelation of a target protease through the two binding sites is a unique feature of ecotin since most serine protease inhibitors interact with their target proteases predominately through their reactive-site loop (31Bode W. Huber R. Eur. J. Biochem. 1992; 204: 433-451Crossref PubMed Scopus (1002) Google Scholar). The bidentate binding scheme utilized by ecotin may allow fine tuning of protease inhibition toward specific targets through protein engineering efforts. Although the catalytic domain of uPA and trypsin are homologous (7Steffens G.J. Gunzler W.A. Otting F. Frankus E. Flohe L. Hoppe-Seyler's Z Physiol. Chem. 1982; 363: 1043-1058Crossref PubMed Scopus (174) Google Scholar) and their substrate specificities are virtually identical, ecotin is a poor inhibitor of uPA proteolytic activity. We attempted to convert ecotin into a potent uPA inhibitor using phage display to aid our understanding of protease-inhibitor recognition and uPA function. It was previously shown that a bovine pancreatic trypsin inhibitor variant with altered specificity and high affinity toward human neutrophil elastase could be isolated from a library of bovine pancreatic trypsin inhibitor variants by phage display technology (32Roberts B.L. Markland W. Ley A.C. Kent R.B. White D.W. Guterman S.K. Ladner R.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2429-2433Crossref PubMed Scopus (193) Google Scholar). This demonstrated the feasibility of studying protease-inhibitor interactions using this technique. Phage display allows the expression of a diverse library of peptides or protein variants on the surface of filamentous M13 bacteriophage (33Smith G.P. Science. 1985; 228: 1315-1517Crossref PubMed Scopus (3063) Google Scholar, 34Parmley S.F. Smith G.P. Gene (Amst.). 1988; 73: 305-318Crossref PubMed Scopus (734) Google Scholar). This in turn allows the isolation of individual phage particles that display desired binding properties by an in vitro selection process. Since the phenotype of each phage is directly linked to its genotype, specific mutations in the displayed peptide or protein that confer a desired function can be readily identified. Using this technology, a number of antigen-antibody interactions have been studied (34Parmley S.F. Smith G.P. Gene (Amst.). 1988; 73: 305-318Crossref PubMed Scopus (734) Google Scholar, 35Hawkins R.E. Russell S.J. Winter G. J. Mol. Biol. 1992; 226: 889-896Crossref PubMed Scopus (466) Google Scholar) as well as hormone-receptor interactions (36Bass S. Greene R. Wells J.A. Proteins. 1990; 8: 309-314Crossref PubMed Scopus (320) Google Scholar, 37Lowman H.B. Wells J.A. J. Mol. Biol. 1993; 234: 564-578Crossref PubMed Scopus (219) Google Scholar), protein-nucleic acid interactions (38Jamieson A.C. Kim S.H. Wells J.A. Biochemistry. 1994; 33: 5689-5695Crossref PubMed Scopus (209) Google Scholar, 39Rebar E.J. Pabo C.O. Science. 1994; 263: 671-673Crossref PubMed Scopus (382) Google Scholar), and inhibitor-protease interactions (32Roberts B.L. Markland W. Ley A.C. Kent R.B. White D.W. Guterman S.K. Ladner R.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2429-2433Crossref PubMed Scopus (193) Google Scholar, 40Pannekoek H. van Meijer M. Schleef R.R. Loskutoff D.J. Barbas C.F. Gene (Amst.). 1993; 128: 135-140Crossref PubMed Scopus (52) Google Scholar). We have shown that the phage display approach can be broadly generalized to other protease inhibitors by displaying ecotin on the surface of phagemid-derived bacteriophage (ecotin phage). Ecotin was chosen because of its unusual ability to inhibit trypsin, chymotrypsin, and elastase (26Chung C.H. Ives H.E. Almeda S. Goldberg A.L. J. Biol. Chem. 1983; 258: 11032-11038Abstract Full Text PDF PubMed Google Scholar), and its unique mechanism of inhibition (30McGrath M.E. Erpel T. Bystroff C. Fletterick R.J. EMBO J. 1994; 13: 1502-1507Crossref PubMed Scopus (71) Google Scholar). Its broad specificity suggests a structural flexibility which would allow modifications via protein engineering to confer novel properties. The crystal structure of ecotin complexed with a variant of rat trypsin was solved recently (30McGrath M.E. Erpel T. Bystroff C. Fletterick R.J. EMBO J. 1994; 13: 1502-1507Crossref PubMed Scopus (71) Google Scholar). Combined with information from the three-dimensional structure, phage display can be used to search designed libraries of ecotin mutants that affect interactions at the inhibitor/protease interface. Variants with high affinity toward a particular target protease can then be readily isolated and characterized. Herein, we report the display of ecotin on the surface of phagemid-derived bacteriophage, and the isolation of mutants with high affinity toward uPA. Enzymes and reagents for molecular cloning were purchased from New England Biolabs and were used following the manufacturer's instructions. The E. coli strain JM101 and the VCSM13 helper phage were from Stratagene. Low molecular weight uPA (LMuPA) was obtained from American Diagnostica. Bovine trypsin was from Sigma. The chromogenic substrate Z-Gly-Pro-Arg-p-nitroanilide used for trypsin kinetics analysis was from Bachem, and the chromogenic substrate Z-γ-Glu(α-t-butoxy)-Gly-Arg-p-nitroanilide (Spectrozyme UK) used for LMuPA kinetics analysis was from American Diagnostica. 4-Methylumbelliferyl p-guanidinobenzoate was from Sigma. α-35S-dATP was from DuPont NEN. Sequenase Version 2.0 sequencing kit was from U. S. Biochemical Corp. Oligonucleotides were synthesized with an Applied Biosystems 391 DNA synthesizer. The phagemid pBSeco-gIII was constructed to produce ecotin on the surface of the surface of filamentous phage. These phage are referred to as ecotin phage. The ecotin expression plasmid pTacTacEcotin (41McGrath M.E. Erpel T. Browner M.F. Fletterick R.J. J. Mol. Biol. 1991; 222: 139-142Crossref PubMed Scopus (25) Google Scholar) was digested with the restriction endonucleases BamHI and HindIII. The resulting DNA fragment encoding the ecotin gene and its signal sequence was ligated to the large fragment of BamHI/HindIII-digested pBluescript to produce pBSecotin. The DNA sequence coding for amino acids 198-406 of gene III of M13 was generated from M13 mp18 DNA using polymerase chain reaction; the forward primer was 5’-GTC ACG AAG CTT CCA TTC GTT TGT GAA TAT CAA GG-3’, and the reverse primer was 5’-GCA CGA AGC TTA AGA CTC CTT ATT ACG CAG TAT G-3’. After HindIII digestion, the polymerase chain reaction product was inserted into a HindIII site at the 3’ end of the ecotin gene of pBSecotin. The stop codon at the COOH terminus of the ecotin gene was then removed, and a Gly-Gly-Gly tether was introduced at the junction of the fusion gene. This was achieved by site-directed mutagenesis (42Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4894) Google Scholar) using the primer 5’-CG GTA GTT CGC GGC GGC GGA GCT GAA AGC GTC CAG-3’. The resulting plasmid construct was named pBSeco-gIII. A pBSeco-gIII mutant in which codons 84 and 85 and the third base pair of codon 83 of the ecotin gene were deleted was constructed to provide control phage that did not express the ecotin gene. This mutant, pBSeco-gIIIΔ, was made by site-directed mutagenesis using the primer 5’-C AGT TCC CCG GTT AGT AC GCC TGC CCG GAT GG-3’. A pBSeco-gIII library with random mutations at codons 84 and 85 of the “reactive-site” loop of ecotin was created by oligonucleotide-directed mutagenesis using the oligonucleotide 5’-C AGT TCC CCG GTT AGT ACT NNS NNS GCC TGC CCG GAT GG-3’ (N = A/C/G/T; S = G/C) as the primer and the uracilated, single-stranded pBSeco-gIII as the template. Also introduced by this primer was a ScaI site, which enabled facile differentiation between native templates and mutant templates. The library of ecotin phage had 1024 possible DNA sequences that resulted in 400 possible protein sequences. For the preparation of pBluescript, pBSeco-gIII, and pBSeco-gIIIΔ bacteriophage, plasmid DNAs were transformed into a male strain (F’) of JM101. A single colony selected on ampicillin plates was grown in 3 ml of 2YT medium (16 g of tryptone, 10 g of yeast extract, 5 g of NaCl/liter) containing 60 μg/ml ampicillin at 37°C for 7 h. The culture was diluted into 30-100 ml of 2YT/ampicillin, grown to A600 = 0.25, and infected with the helper phage VCSM13 at a multiplicity of infection of approximately 100 helper phage per cell. The infected culture was allowed to grow at 37°C with shaking for approximately 12 h. Phage particles were harvested by precipitation with 5% polyethylene glycol and resuspended in 1 ml of TBS buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4). Phage titers typically ranged from 5 ×1010 to 2× 1011 cfu/ml culture. For library phage preparation, the mutagenesis reaction mixture was ethanol-precipitated, redissolved in water, electroporated into F’ JM101, and plated on 150-mm LB/ampicillin plates. Cells from the plates were recovered in 5 ml of LB/ampicillin and diluted in 50 ml of 2YT/ampicillin to an A600 = 0.25, and then infected with VCSM13 helper phage. The infected culture was grown for 12 h at 37°C with shaking, and the phage were harvested as described above. Approximately 5 × 1010 cfu were loaded in duplicate onto a single 1% agarose gel with 25 mM Tris, 250 mM glycine (pH 8.6) as the running buffer (43Corey D.R. Shiau A.K. Yang Q. Janowski B.A. Craik C.S. Gene (Amst.). 1993; 128: 129-134Crossref PubMed Scopus (84) Google Scholar). The gel was electrophoresed at 6 mA constant current for 16 h. One set of samples was transblotted onto a nitrocellulose filter. The filter was immunostained for ecotin by allowing it to react with rabbit anti-ecotin antibodies followed by reaction with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies. The other set of samples were denatured by soaking the gel in 0.5 N NaOH for 4 h, washed by soaking in water for 4-8 h, and stained with ethidium bromide. pBSeco-gIII and pBSeco-gIIIΔ phage were suspended in trypsin assay buffer (50 mM Tris-HCl, 100 mM NaCl, 20 mM CaCl2, pH 8.0) and adjusted to 1.5 × 101313 cfu/ml. Various volumes of phage solution were incubated with 0.5 nM trypsin in a total volume of 125 μl of trypsin assay buffer, 0.01% Tween 80 in a 96-well microtiter plate at room temperature for 20 min. After adding 125 μl of 0.1 mM substrate Z-Gly-Pro-Arg-p-nitroanilide, the residual trypsin activity was measured by monitoring the increase of optical density at 405 nm. Polystyrene Petri dishes (35 mm, Falcon) were coated with 1 ml of 10 μg/ml bovine trypsin or LMuPA in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM K2HPO4, pH 7.5) overnight, and excess binding sites were blocked with 5% non-fat dry milk solution for 2 h. For control experiments, Petri dishes were coated with 5% non-fat dry milk solution. Phage were added to the dishes in buffer containing 1 ml of phosphate-buffered saline, 0.5% Tween 20 and incubated overnight with gentle agitation at room temperature. Solutions containing the phagemid were then removed and the dishes were washed 9 times with 5 ml of phosphate-buffered saline/Tween 20. Each wash was approximately 1 min. Bound phage were serially eluted by incubation with 1 ml of 0.1 N HCl/glycine solution (pH 2.2) with gentle shaking for 15 min at room temperature. Three elutions were performed. The eluates were neutralized with 185 μl of 1 M Tris-HCl (pH 8.8). For biopanning against immobilized trypsin, a mixture of pBSeco-gIII phage (1.1 × 108 cfu) and pBluescript phage (2.8 × 108cfu) was used. An aliquot of the appropriately diluted solution of each wash and elution was used to infect 100 μl of saturated JM101 cells. After incubation for 15 min at 37°C, the infected cells were plated on LB/ampicillin plates containing IPTG and 5-bromo-4-chloro-3-indolyl β-D-galactoside. The cfu ratio of pBSeco-gIII phage to pBluescript phage was calculated by the number of white and blue colonies, respectively. For biopanning against immobilized LMuPA, a mixture of 2.5 × 1010 cfu library phage were used. Phage from the third elution were amplified for the next cycle of panning. Ecotin and ecotin mutants were produced in bacteria from the expression vector pTacTacEcotin (41McGrath M.E. Erpel T. Browner M.F. Fletterick R.J. J. Mol. Biol. 1991; 222: 139-142Crossref PubMed Scopus (25) Google Scholar). The expression and purification procedures were as follows. JM101 cells were freshly transformed with expression plasmid DNA. A single colony selected from ampicillin plates was used to inoculate 3 ml of LB containing 60 μg/ml ampicillin. The cultures were grown at 37°C for 9 h and diluted to 1 liter of LB/ampicillin. Following growth at 37°C for 1 h, IPTG was added to the cultures to a final concentration of 0.2 mM, and continued to grow for 12 h at for 37°C. Cells were harvested and treated with lysozyme in a solution containing 25% sucrose, 10 mM Tris-HCl (pH 8.0). The periplasmic fraction was dialyzed against 10 mM sodium citrate (pH 2.8). Following the dialysis, the supernatant was adjusted to pH 7.4 with 1 M Tris-HCl (pH 8.0), and to 0.3 M NaCl. The solution was heated in boiling water for 10 min, and then cooled to room temperature. The precipitate was removed by centrifugation, and the supernatant was dialyzed against water overnight at 4°C. The solution containing the ecotin was loaded onto a Vydac C4 reverse-phase high performance liquid chromatography column (2.2 × 25 cm) which was equilibrated with 0.1% trifluoroacetic acid. The column was washed and then eluted with a linear gradient of 34-37% acetonitrile, 0.1% trifluoroacetic acid at a flow rate of 10 ml/min over 30 min. Fractions were analyzed with SDS-polyacrylamide gel electrophoresis (44Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar), and the ones containing pure ecotin were pooled and lyophilized. Purified ecotin was redissolved in buffer containing 10 mM Tris-HCl (pH 7.4) and stored at 4°C. The concentrations of ecotin and ecotin mutants were determined using a calculated molar extinction coefficient (45Gill S.C. von Hippel H.P. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5034) Google Scholar) of 2.2 × 104 cm-1M-1 and were in good agreement with that from the Bradford assay (data not shown) (46Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar). LMuPA was titrated with 4-methylumbelliferyl p-guanidinobenzoate to obtain an accurate concentration of enzyme active sites. Various concentrations of ecotin or ecotin mutants were incubated with human LMuPA in a total volume of 990 μl of buffer containing 50 mM NaCl, 50 mM Tris-HCl (pH 8.7), 0.01% Tween 80. The final concentrations of LMuPA used for the determination of equilibrium dissociation constants, Ki, of ecotin or ecotin mutants were as follows: 0.5 nM (M84R/M85R); 1.0 nM (M84R, M84K, and M84R/M85K); 7.2 nM (wild-type). The final concentrations of ecotin mutants ranged from 0.6 to 40 nM, and the concentrations of wild-type ecotin ranged from 1.25 to 50 μM. Following a 30-min incubation at room temperature to reach equilibrium, 10 μl of 10 mM substrate Z-γ-Glu (α-t-butoxy)-Gly-Arg-p-nitroanilide was added and the rate of p-nitroaniline formation was measured by monitoring the change of absorption at 410 nm in a 10-min period. The data were fit to the equation derived for kinetics of reversible tight-binding inhibitors (47Morrison J.F. Biochim. Biophys. Acta. 1969; 185: 269-286Crossref PubMed Scopus (715) Google Scholar, 48Williams J.W. Morrison J.F. Methods. Enzymol. 1979; 63: 437-467Crossref PubMed Scopus (658) Google Scholar) by nonlinear regression analysis, and the values f" @default.
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• W2041504987 title "Isolation of a High Affinity Inhibitor of Urokinase-type Plasminogen Activator by Phage Display of Ecotin" @default.
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• W2041504987 doi "https://doi.org/10.1074/jbc.270.20.12250" @default.
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