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• W2048428214 abstract "Type 1 plasminogen activator inhibitor (PAI-1) is a key regulator of the fibrinolytic cascade that is stored in a rapidly releasable form within platelet α-granules. To identify proteins that may participate in the targeting or storage of this potent inhibitor, this report investigates the applicability of utilizing filamentous bacteriophages to display proteins expressed by cells containing a regulated secretory pathway and their enrichment based upon an interaction with PAI-1. For this purpose, RNA was extracted from AtT-20 cells (i.e. a classical model cell system for intracellular protein sorting), reverse transcribed, amplified using polymerase chain reaction primers containing internal restriction sites, and cloned into the phagemid pCOMB3H for expression as fusion constructs with the bacteriophage gene III protein. Escherichia coli was transformed with the phagemids and infected with VCSM13 helper phage, and the resulting AtT-20 cDNA-bacteriophage library was enriched by panning against solid- and solution-phase PAI-1. The enriched cDNA library was subcloned into a prokaryotic expression vector system that replaces the gene III protein with a decapeptide tag for immunologic quantitation. One novel cDNA clone (i.e. A-61), which preferentially recognized solution-phase PAI-1 and reacted positively with antibodies derived from a rabbit immunized with α-granules, was subcloned into the prokaryotic expression vector pTrcHis to create a construct containing an N-terminal six-histidine purification tag. This construct was expressed in E. coli, purified by nickel-chelate chromatography followed by preparative SDS-polyacrylamide gel electrophoresis, and utilized for the generation of polyclonal antibodies. Immunoblotting analysis employing antibodies against the purified A-61 construct revealed a 23-kDa protein present in the regulated secretory pathway of AtT-20 cells. The 23-kDa molecule was purified from media conditioned by AtT-20 cells by ion exchange chromatography on DEAE-Sephacel, molecular sieve chromatography on Sephacryl S-100, chromatofocusing on Polybuffer exchanger 94, and affinity chromatography on PAI-1-Sepharose. N-terminal amino acid sequencing of a 16-kDa Lys-C proteolytic fragment of the 23-kDa storage granule protein was employed to confirm its identity with the cDNA sequence of clone A-61. These data indicate that phage display of cDNA libraries fused to the C-terminal region of the gene III protein and their enrichment via an interaction with a target molecule can be utilized to define other proteins present within a particular cellular pathway. Type 1 plasminogen activator inhibitor (PAI-1) is a key regulator of the fibrinolytic cascade that is stored in a rapidly releasable form within platelet α-granules. To identify proteins that may participate in the targeting or storage of this potent inhibitor, this report investigates the applicability of utilizing filamentous bacteriophages to display proteins expressed by cells containing a regulated secretory pathway and their enrichment based upon an interaction with PAI-1. For this purpose, RNA was extracted from AtT-20 cells (i.e. a classical model cell system for intracellular protein sorting), reverse transcribed, amplified using polymerase chain reaction primers containing internal restriction sites, and cloned into the phagemid pCOMB3H for expression as fusion constructs with the bacteriophage gene III protein. Escherichia coli was transformed with the phagemids and infected with VCSM13 helper phage, and the resulting AtT-20 cDNA-bacteriophage library was enriched by panning against solid- and solution-phase PAI-1. The enriched cDNA library was subcloned into a prokaryotic expression vector system that replaces the gene III protein with a decapeptide tag for immunologic quantitation. One novel cDNA clone (i.e. A-61), which preferentially recognized solution-phase PAI-1 and reacted positively with antibodies derived from a rabbit immunized with α-granules, was subcloned into the prokaryotic expression vector pTrcHis to create a construct containing an N-terminal six-histidine purification tag. This construct was expressed in E. coli, purified by nickel-chelate chromatography followed by preparative SDS-polyacrylamide gel electrophoresis, and utilized for the generation of polyclonal antibodies. Immunoblotting analysis employing antibodies against the purified A-61 construct revealed a 23-kDa protein present in the regulated secretory pathway of AtT-20 cells. The 23-kDa molecule was purified from media conditioned by AtT-20 cells by ion exchange chromatography on DEAE-Sephacel, molecular sieve chromatography on Sephacryl S-100, chromatofocusing on Polybuffer exchanger 94, and affinity chromatography on PAI-1-Sepharose. N-terminal amino acid sequencing of a 16-kDa Lys-C proteolytic fragment of the 23-kDa storage granule protein was employed to confirm its identity with the cDNA sequence of clone A-61. These data indicate that phage display of cDNA libraries fused to the C-terminal region of the gene III protein and their enrichment via an interaction with a target molecule can be utilized to define other proteins present within a particular cellular pathway. Type-1 plasminogen activator inhibitor (PAI-1) 1The abbreviations used are: PAI-1type 1 plasminogen activator inhibitorserpinserine protease inhibitorBSAbovine serum albuminPBSphosphate-buffered salineSGP-2323-kDa storage granule proteinPAGEpolyacrylamide gel electrophoresisTBSTris-buffered salineACTHadrenocorticotropic hormoneMops4-morpholinepropanesulfonic acid. is the primary physiological inhibitor of vascular tissue-type plasminogen activator (for reviews, see Refs. 1Van Meijer M. Pannekoek H. Fibrinolysis. 1995; 9: 263-276Crossref Scopus (142) Google Scholar and 2Reilly T.M. Mousa S.A. Seetharam R. Racanelli A.L. Blood Coagul. & Fibrinolysis. 1994; 5: 73-81Crossref PubMed Scopus (19) Google Scholar). The role of PAI-1 as a key physiological regulator of the fibrinolytic system is supported by the correlation of bleeding disorders in a number of patients that have a deficiency in blood PAI-1 activity (3Schleef R.R. Higgins D.L. Pillemer E. Levitt J.J. J. Clin. Invest. 1989; 83: 1747-1752Crossref PubMed Scopus (182) Google Scholar, 4Dieval J. Nguyen G. Gross S. Delobel J. Kruithof E.K.O. Blood. 1991; 73: 528-532Crossref Google Scholar, 5Lee M.H. Vosburgh E. Anderson K. McDonagh J. Blood. 1993; 81: 2357-2362Crossref PubMed Google Scholar, 6Fay W.P. Shapiro A.D. Shih J.L. Schleef R.R. Ginsburg D. N. Engl. J. Med. 1992; 327: 1729-1733Crossref PubMed Scopus (224) Google Scholar). Sequence analysis of the cDNA encoding PAI-1 has led to the classification of this inhibitor in the serpin superfamily (1Van Meijer M. Pannekoek H. Fibrinolysis. 1995; 9: 263-276Crossref Scopus (142) Google Scholar, 2Reilly T.M. Mousa S.A. Seetharam R. Racanelli A.L. Blood Coagul. & Fibrinolysis. 1994; 5: 73-81Crossref PubMed Scopus (19) Google Scholar). This inhibitor is produced as a Mr 50,000 glycoprotein by a wide variety of cells and is present in blood either at low concentrations in plasma or in a large storage pool within platelets (7Erickson L.A. Ginsberg M.H. Loskutoff D.J. J. Clin. Invest. 1984; 74: 1465-1472Crossref PubMed Scopus (242) Google Scholar, 8Booth N.A. Croll A. Bennett B. Fibrinolysis. 1990; 4: 138-140Crossref Scopus (29) Google Scholar, 9Declerck P.J. Alessi M. Verstreken M. Kruithof E.K.O. Juhan-Vague I. Collen D. Blood. 1988; 71: 220-225Crossref PubMed Google Scholar, 10Kruithof E.K.O. Nicolosa G. Bachmann F.W. Blood. 1987; 70: 1645-1653Crossref PubMed Google Scholar, 11Booth N.A. Anderson J.A. Bennett B. J. Clin. Pathol. 1985; 38: 825-830Crossref PubMed Scopus (57) Google Scholar, 12Simpson A.J. Booth N.A. Moore N.R. Bennett B. Br. J. Haematol. 1990; 75: 543-548Crossref PubMed Scopus (38) Google Scholar, 13Sprengers E.D. Akkerman J.W.N. Jansen B.G. Thromb. Haemostasis. 1986; 55: 325-329Crossref PubMed Scopus (76) Google Scholar, 14Kruithof E.K.O. Tran-Thang C. Bachmann F.W. Thromb. Haemostasis. 1986; 55: 201-205Crossref PubMed Scopus (112) Google Scholar, 15Booth N.A. Simpson A.J. Croll A. Bennett B. Macgregor I.R. Br. J. Haematol. 1988; 70: 327-333Crossref PubMed Scopus (253) Google Scholar). The presence of PAI-1 mRNA and antigen in megakaryocytes (16Simpson A.J. Booth N.A. Moore N.R. Bennett B. J. Clin. Pathol. 1991; 44: 139-143Crossref PubMed Scopus (126) Google Scholar, 17Konkle B.A. Schick P.K. He X. Liu R.J. Mazur E.M. Arterioscler. Thromb. 1993; 13: 669-674Crossref PubMed Google Scholar, 18Alessi M.C. Chomiki N. Berthier R. Schweitzer A. Fossat C. Juhan-Vague I. Thromb. Haemostasis. 1994; 72: 931-936Crossref PubMed Scopus (27) Google Scholar), the hemopoietic precursor of platelets, suggests that PAI-1 may be deposited into storage organelles (i.e. α-granules) during the maturation of these cells. type 1 plasminogen activator inhibitor serine protease inhibitor bovine serum albumin phosphate-buffered saline 23-kDa storage granule protein polyacrylamide gel electrophoresis Tris-buffered saline adrenocorticotropic hormone 4-morpholinepropanesulfonic acid. Current information indicates that PAI-1 is synthesized in an active form, but it is rapidly converted into an inactive form at 37°C with a half-life of approximately 1 h (for a review, see Ref. 1Van Meijer M. Pannekoek H. Fibrinolysis. 1995; 9: 263-276Crossref Scopus (142) Google Scholar). The conformation of PAI-1 resulting from inactivation at 37°C is commonly referred to as latent PAI-1 because inhibitory activity can be detected following treatment with denaturants or negatively charged phospholipids (for reviews, see Refs. 1Van Meijer M. Pannekoek H. Fibrinolysis. 1995; 9: 263-276Crossref Scopus (142) Google Scholar and 2Reilly T.M. Mousa S.A. Seetharam R. Racanelli A.L. Blood Coagul. & Fibrinolysis. 1994; 5: 73-81Crossref PubMed Scopus (19) Google Scholar). In light of the observation that platelets possess low biosynthetic capabilities (19Bithell T.C. Bithell T.C. Clinical Hematology. Lea & Febiger, Philadelphia1993: 511-539Google Scholar), it is not unexpected that the majority of PAI-1 is present within platelets in a latent form. Although vitronectin is known to be capable of increasing by 2-fold the half-life of PAI-1 activity in solution (37°C) (for a review, see Ref. 20Preissner K.T. Jenne D. Thromb. Haemostasis. 1991; 66: 123-132Crossref PubMed Scopus (85) Google Scholar), recent data from our group (21Lang I.M. Schleef R.R. J. Biol. Chem. 1996; 271: 2754-2761Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) indicate that complexes between vitronectin and PAI-1 are not present in nonactivated platelets. Therefore, little information exists on the proteins that interact and stabilize PAI-1 stored within platelets that have a mean life span of 9-12 days in the circulation (19Bithell T.C. Bithell T.C. Clinical Hematology. Lea & Febiger, Philadelphia1993: 511-539Google Scholar). Two distinct pathways are known to be responsible for the secretion of proteins from eukaryotic cells (for reviews, see Refs. 22Burgess T.L. Kelly R.B. Annu. Rev. Cell Biol. 1987; 3: 243-293Crossref PubMed Scopus (743) Google Scholar and 23Rindler M.J. Curr. Opin. Cell Biol. 1992; 4: 616-622Crossref PubMed Scopus (25) Google Scholar). The “constitutive” pathway externalizes proteins rapidly using post-Golgi vesicles and does not require an external stimulus for release of a compound into the extracellular milieu (22Burgess T.L. Kelly R.B. Annu. Rev. Cell Biol. 1987; 3: 243-293Crossref PubMed Scopus (743) Google Scholar, 23Rindler M.J. Curr. Opin. Cell Biol. 1992; 4: 616-622Crossref PubMed Scopus (25) Google Scholar). In the “regulated” pathway, proteins are stored in secretory granules until the cells are stimulated to secrete in response to the appropriate stimuli (22Burgess T.L. Kelly R.B. Annu. Rev. Cell Biol. 1987; 3: 243-293Crossref PubMed Scopus (743) Google Scholar, 23Rindler M.J. Curr. Opin. Cell Biol. 1992; 4: 616-622Crossref PubMed Scopus (25) Google Scholar). A number of tumor-derived cell lines exhibit both a constitutive and a regulated secretory pathway, and these cell lines have been used as in vitro model cell systems for analyzing the processing of proteins into these two pathways (22Burgess T.L. Kelly R.B. Annu. Rev. Cell Biol. 1987; 3: 243-293Crossref PubMed Scopus (743) Google Scholar, 23Rindler M.J. Curr. Opin. Cell Biol. 1992; 4: 616-622Crossref PubMed Scopus (25) Google Scholar). A classical system is the mouse pituitary tumor cell line, AtT-20, that has been shown to divert a majority of the endogenously synthesized adrenocorticotropic hormone (ACTH) into the regulated storage pathway (24Gumbiner B. Kelly R.B. Cell. 1982; 28: 51-59Abstract Full Text PDF PubMed Scopus (213) Google Scholar, 25Burgess T.L. Craik C.S. Kelly R.B. J. Cell Biol. 1985; 101: 639-645Crossref PubMed Scopus (44) Google Scholar). Treatment of AtT-20 cells with the appropriate secretagogue (e.g. 8-Br-cyclic AMP) results in release of the contents of the secretory granule (24Gumbiner B. Kelly R.B. Cell. 1982; 28: 51-59Abstract Full Text PDF PubMed Scopus (213) Google Scholar, 26Moore H.H. Walker M.D. Lee F. Kelly R.B. Cell. 1983; 35: 531-538Abstract Full Text PDF PubMed Scopus (228) Google Scholar). These cells have been shown to have the capacity, after transfection with the appropriate DNA, to package heterologous peptide hormones and enzymes into the regulated secretory pathway. For example, proinsulin (26Moore H.H. Walker M.D. Lee F. Kelly R.B. Cell. 1983; 35: 531-538Abstract Full Text PDF PubMed Scopus (228) Google Scholar), trypsinogen (25Burgess T.L. Craik C.S. Kelly R.B. J. Cell Biol. 1985; 101: 639-645Crossref PubMed Scopus (44) Google Scholar, 27Burgess T.L. Craik C.S. Matsuuchi L. Kelly R.B. J. Cell Biol. 1987; 105: 659-668Crossref PubMed Scopus (44) Google Scholar), human growth hormone (28Moore H.H. Kelly R.B. Nature. 1986; 321: 443-446Crossref PubMed Scopus (91) Google Scholar), and peptidylglycine α-amidating monooxygenase (29Milgram S.L. Johnson R.C. Mains R.E. J. Cell Biol. 1992; 117: 717-728Crossref PubMed Scopus (103) Google Scholar) are transported to the regulated pathway with a similar efficiency as the endogenous hormone, ACTH. This cell line has also proven useful for investigating the packaging of two proteins stored in both endothelial cells and platelets (i.e. P-selectin (30Koedam J.A. Cramer E.M. Briend E. Furie B. Furie B.C. Wagner D.D. J. Cell Biol. 1992; 116: 617-625Crossref PubMed Scopus (103) Google Scholar, 31Disdier M. Morrissey J.H. Fugate R.D. Bainton D.F. McEver R.P. Mol. Biol. Cell. 1992; 3: 309-321Crossref PubMed Scopus (125) Google Scholar) and von Willebrand's factor (32Wagner D.D. Saffaripour S. Bonfanti R. Sadler J.E. Cramer E.M. Chapman B. Mayades T.N. Cell. 1991; 64: 403-413Abstract Full Text PDF PubMed Scopus (208) Google Scholar)). Furthermore, transfection experiments with full-length PAI-1 cDNA demonstrated that this inhibitor is also packaged into AtT-20 dense core storage granules (33Gombau L. Schleef R.R. J. Biol. Chem. 1994; 269: 3875-3880Abstract Full Text PDF PubMed Google Scholar). Analysis of PAI-1 within the isolated storage granules has revealed (i) the presence of both active and latent PAI-1 in a ratio comparable with the situation in human platelets and (ii) that PAI-1 activity is stabilized within the secretory granules (33Gombau L. Schleef R.R. J. Biol. Chem. 1994; 269: 3875-3880Abstract Full Text PDF PubMed Google Scholar). Taken together, this cell line appears to be a useful model system for identification of proteins within the regulated secretory pathway that interact with PAI-1 and potentially participate in its targeting or stabilization. The ability of filamentous bacteriophages to display proteins on their surface has been used for the generation of libraries of recombinant antibody fragments (for reviews, see Refs. 34Lerner R.A. Kang A.S. Bain J.D. Burton D.R. Barbas C.F. Science. 1992; 258: 1313-1314Crossref PubMed Scopus (87) Google Scholar, 35Winter G. Griffiths A.D. Hawkins R.E. Hoogenboom H.R. Annu. Rev. Immunol. 1994; 12: 433-455Crossref PubMed Scopus (1374) Google Scholar, 36Burton D.R. Barbas C.F.I. Adv. Immunol. 1994; 57: 191-280Crossref PubMed Google Scholar) and peptide libraries (for reviews, see Refs. 37Scott J.K. Trends Biochem. Sci. 1992; 17: 241-245Abstract Full Text PDF PubMed Scopus (107) Google Scholar and 38Lane D.P. Stephen C.W. Curr. Opin. Immunol. 1993; 5: 268-271Crossref PubMed Scopus (50) Google Scholar). Antibody fragments or peptides are expressed as fusion proteins with the bacteriophage's gene III or gene VIII surface protein, and the characteristics of the surface-expressed molecule (e.g. affinity or interaction with a ligand) can be used as a means to enrich the phage (34Lerner R.A. Kang A.S. Bain J.D. Burton D.R. Barbas C.F. Science. 1992; 258: 1313-1314Crossref PubMed Scopus (87) Google Scholar, 35Winter G. Griffiths A.D. Hawkins R.E. Hoogenboom H.R. Annu. Rev. Immunol. 1994; 12: 433-455Crossref PubMed Scopus (1374) Google Scholar, 36Burton D.R. Barbas C.F.I. Adv. Immunol. 1994; 57: 191-280Crossref PubMed Google Scholar, 37Scott J.K. Trends Biochem. Sci. 1992; 17: 241-245Abstract Full Text PDF PubMed Scopus (107) Google Scholar, 38Lane D.P. Stephen C.W. Curr. Opin. Immunol. 1993; 5: 268-271Crossref PubMed Scopus (50) Google Scholar). In this system, the cDNA encoding the surface-expressed protein/peptide is contained within the bacteriophage's genetic material, thus permitting the rapid identification and cloning of a molecule. This study was initiated to investigate the applicability of this system to identify proteins that potentially interact with PAI-1 within the regulated secretory pathway. In this report, we describe the construction of an AtT-20 cDNA-bacteriophage library and its enrichment based upon its interaction with PAI-1 to identify a novel 23-kDa protein that is present within the regulated secretory pathway. AtT-20 cells, DAMI cells (a cell line established from an individual with megakaryoblastic leukemia (39Greenberg S.M. Rosenthal D.S. Greeley T.A. Tantravahi R. Handin R.I. Blood. 1988; 72: 1968-1977Crossref PubMed Google Scholar)), and a transformed human fibroblast cell line (SV40 WI38 VA13 2RA) were obtained from the American Tissue Culture Collection (Rockville, MD). The cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Life Technologies, Inc.) in the presence of either a 5% C02 (DAMI cells and transformed fibroblasts) or 15% CO2 atmosphere (AtT-20 cells (33Gombau L. Schleef R.R. J. Biol. Chem. 1994; 269: 3875-3880Abstract Full Text PDF PubMed Google Scholar)). Native PAI-1 was purified from the media conditioned by transformed human lung fibroblasts as described previously (33Gombau L. Schleef R.R. J. Biol. Chem. 1994; 269: 3875-3880Abstract Full Text PDF PubMed Google Scholar, 40Schleef R.R. Loskutoff D.J. Podor T.J. J. Cell Biol. 1991; 113: 1413-1423Crossref PubMed Scopus (33) Google Scholar). Antibodies to PAI-1 were raised in New Zealand rabbits and affinity-purified on Sepharose-PAI-1 columns as described previously (33Gombau L. Schleef R.R. J. Biol. Chem. 1994; 269: 3875-3880Abstract Full Text PDF PubMed Google Scholar). Human platelets were isolated and utilized for the preparation of α-granules as described previously (21Lang I.M. Schleef R.R. J. Biol. Chem. 1996; 271: 2754-2761Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Briefly, platelets were diluted in homogenization buffer (108 mM NaCl, 38 mM KCl, 1.7 mM NaHCO3, 21.2 mM sodium citrate, 27.8 mMD-glucose, 1.1 mM MgCl2, 1 mM theophyllin, pH 6.5) to a final concentration of 109/ml and sonicated using an Astrason Ultrasonic processor XL (Heat Systems Inc., Farmingdale, NY) five times (4°C, 3 s of sonication on setting 2 followed by a 15-s pause between each sonication). Samples were centrifuged (15 min, 2,000 × g); the membrane/organelle/cytosol-containing supernatants were pooled and mixed 1:1 with 40% metrizamide solution (Accurate Chemical and Scientific Co., Westbury, NY); and this mixture was layered on top of a two-step gradient consisting of 1 ml of 35% metrizamide underlayered with 0.5 ml of 38% metrizamide. Following centrifugation (1 h, 4°C, 100,000 × g), the α-granules were harvested from the 20-35% metrizamide interface. This preparation was mixed with an equal volume of 90% stock isotonic Percoll (1 ml of 1.5 M NaCl with 9 ml of Percoll; Pharmacia Biotech, Inc.) and ultracentrifuged (4°C, 30 min, 20,000 × g). The α-granules were recovered as an opaque band at a density of 1.06-1.1 g/liter. Rabbit antibodies directed against α-granules were prepared by immunizing a New Zealand White rabbit with 0.5 ml of isolated α-granules (20 mg of protein/injection) dissolved in Freund's adjuvant over a period of 6 months utilizing standard techniques. The IgG fraction of the antiserum was isolated by ammonium sulfate precipitation and affinity-purified utilizing CNBr-activated Sepharose beads that were coupled to α-granule proteins according to the manufacturer's instructions (Pharmacia). AtT-20 and DAMI cells (1.5 × 108 cells, 106 cells/ml) were harvested separately into guanidine thiocyanate followed by extraction of total RNA according to the procedures described by Chomczynski and Sacchi (41Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63019) Google Scholar) and as detailed previously (42Schleef R.R. Bevilacqua M.P. Sawdey M. Gimbrone Jr., M.A. Loskutoff D.J. J. Biol. Chem. 1988; 263: 5797-5803Abstract Full Text PDF PubMed Google Scholar). AtT-20 RNA (15 μg) was reverse transcribed utilizing the commercially available First Strand cDNA synthesis kit (Boehringer Mannheim) and oligo(dt)15 primers. AtT-20 cell cDNA was amplified in a Perkin-Elmer 9600 thermal cycler utilizing the forward primer (5′-CAGTCGCTCGAGRNNATG-3′) that contained an internal XhoI site (underlined) and, in separate reactions, reverse primers (i.e. 5′-TGGGCAACTAGTGTANNNNNN-3′; 5′-TGGGCAACTAGTCCANNNNNN-3′) that contained an internal SpeI site (underlined). The following protocol, known as “touchdown” PCR, was utilized to derive specific PCR products: 5 min at 94°C, 20 cycles consisting of 30 s at 94°C, 45 s at 53°C (lowering temperature 0.5°C each cycle), 3 min at 70°C, followed by 5 cycles utilizing 30 s at 94°C, 45 s at 43°C, and 3 min at 70°C. PCR products from each library were pooled and subjected to agarose gel electrophoresis, and the region between 3000 and 200 base pairs was electrophoretically eluted from the agarose gel. This material was digested with an excess amount of restriction enzymes SpeI and XhoI (50 and 200 units/μg DNA, respectively) and ligated into the vector pCOMB3H, a variant of the phagemid pComb3 (43Barbas C.F. Kang A.S. Lerner R.A. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7978-7982Crossref PubMed Scopus (1027) Google Scholar). The final library consisted of 1.5 × 106 clones. Phagemids were transformed into Escherichia coli XL1-Blue cells and grown in super broth medium (SB; 30 g/liter tryptone, 20 g/liter yeast extract, and 10 g/liter Mops, pH 7) at 37°C supplemented with tetracyclin (10 μg/ml) and carbenicillin (50 μg/ml). Cultures were grown to an A600 of 0.8, infected with VCSM13 helper phage (4 × 1011 plaque-forming units/ml), and grown 2 additional h. Kanamycin was added (70 μg/ml), and the culture was incubated overnight. Phage were isolated from liquid culture by polyethylene glycol 8000 and NaCl precipitation (43Barbas C.F. Kang A.S. Lerner R.A. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7978-7982Crossref PubMed Scopus (1027) Google Scholar). Phage pellets were suspended in Tris-buffered saline (TBS; 50 mM Tris-HCl, pH 7.5, 150 mM NaCl), 1% bovine serum albumin (BSA). Enrichment of phages was performed on microtiter plates (Costar 3690) under the following conditions. Wells were precoated with 1 μg of PAI-1 or 2 μg of affinity-purified rabbit antibodies to PAI-1. Wells were washed twice with water and blocked for 1 h with 3% (w/v) BSA in TBS. Wells coated with PAI-1 were directly incubated (2 h, 37°C) with 50 μl of mixed phage (typically 1011 to 1012 colony-forming units). Alternatively, 50 μl of phage (>108 phage) were incubated (30 min, 37°C) with 20 ng of PAI-1 (final concentration) followed by incubation (2 h, 37°C) on polyclonal anti-PAI-1-coated wells. Following incubation, the wells were washed once (first round of panning), 5 times (second round of panning), or 10 times (third to sixth round of panning) with TBS, 0.05% Tween solution. After a final rinse in distilled water, the adherent phage were eluted by incubation (10 min, 22°C) with 50 μl of elution buffer (0.1 M HCl, adjusted to pH 2.2 with glycine) containing 1 mg/ml BSA. The eluant was removed and neutralized with 3 μl of 2 M Tris base. The initial phage input was determined by titering on selective plates. The final phage output was determined by infecting 1 ml of logarithmic phase XLI-Blue cells with the neutralized eluant for 15 min at room temperature and plating equal aliquots on carbenicillin plates. Phagemid DNA from the panned library was isolated, digested with SfiI, and ligated into the arabinose-inducible expression vector pAraHA (43Barbas C.F. Kang A.S. Lerner R.A. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7978-7982Crossref PubMed Scopus (1027) Google Scholar, 44Lang I.M. Barbas III, C.F. Schleef R.R. Gene (Amst.). 1996; 172: 295-298Crossref PubMed Scopus (40) Google Scholar). The ligation mixture was transformed into E. coli DH12S cells and grown overnight in SB containing 30 μg/ml chloramphenicol. Single colonies of AtT-20 cDNA/pAraHA in E. coli were picked, grown for 8 h, and induced by incubation (30°C, 16 h) with 1% arabinose. The bacteria were harvested, lysed into TBS containing 4 μM phenylmethylsulfonyl fluoride (final concentration; Sigma) by four freeze/thawing cycles, and centrifuged, and the cell-free supernatants were incubated (1.5 h, 37°C) in microtiter wells coated with PAI-1 (1 μg/well) or affinity-purified rabbit antibodies against α-granules (1 μg/well). Alternatively, the bacterial lysates were incubated (0.5 h, 37°C) with 20 ng of soluble PAI-1 followed by incubation (1 h, 37°C) of the mixture in wells coated with affinity-purified rabbit antibodies against PAI-1. Controls included wells coated with 1 μg/ml of BSA or normal rabbit IgG. After washing, bound protein was detected by incubating the washed wells with alkaline phosphatase-labeled antidecapeptide (mouse monoclonal anti-YPYDVPDYAS (45Huse W.D. Sastry L. Iverson S.A. Kang A.S. Alting-Mees M. Burton D.R. Benkovic S.J. Lerner R.A. Science. 1989; 246: 1275-1281Crossref PubMed Scopus (685) Google Scholar), 1:500 dilution, 50 μl/well, 2 h, 37°C) followed by kinetic measurement of the resulting color change at 405 nm after the addition of the substrate para-nitrophenylphosphate. The cDNA inserts were sequenced from both strands using the dideoxytermination method (46Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992Google Scholar) coupled with primers synthesized according to the sequences that are either 5′ or 3′ of the SfiI site in the vector pAraHA (43Barbas C.F. Kang A.S. Lerner R.A. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7978-7982Crossref PubMed Scopus (1027) Google Scholar). RNA (10 μg/lane) from AtT-20 and DAMI cells was separated by denaturing electrophoresis in formaldehyde-containing 1% agarose gels and transferred to Hybond-N nylon membranes (Amersham Corp.) as described previously (42Schleef R.R. Bevilacqua M.P. Sawdey M. Gimbrone Jr., M.A. Loskutoff D.J. J. Biol. Chem. 1988; 263: 5797-5803Abstract Full Text PDF PubMed Google Scholar). The cDNA sequence encoding clone A-61 was labeled with [32P]dCTP by random priming using the DECAprime II DNA labeling kit (Ambion Inc., Austin, TX) as described by the manufacturer. The labeled probe was purified using Sephadex G-50 minispin columns (Worthington) resulting in a specific activity of 109 cpm/μg. Hybridization of the labeled probe to the nylon membrane was performed in 5 × SSPE, 5 × Denhardt's solution, 0.5% SDS, and 50 μg/ml fresh denatured salmon sperm DNA (Life Technologies, Inc.) for 15 h at 65°C followed by washing in 0.1 × SSC, 0.1% SDS at 60°C. Hybridization to a 32P-labeled 2.0-kilobase pair human β-actin probe (Clontech) was used to confirm approximate equal loading in all lanes. The blot was exposed for 3 days to Kodak XAR autoradiographic film. SDS-PAGE was performed according to the procedures described by Laemmli (47Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206276) Google Scholar). Gels were processed either by silver staining as described previously (48Lang I.M. Marsh J.J. Moser K.M. Schleef R.R. Blood. 1992; 80: 2269-2274Crossref PubMed Google Scholar), or the electrophoresed proteins were transferred to nitrocellulose as described previously (42Schleef R.R. Bevilacqua M.P. Sawdey M. Gimbrone Jr., M.A. Loskutoff D.J. J. Biol. Chem. 1988; 263: 5797-5803Abstract Full Text PDF PubMed Google Scholar, 49Mimuro J. Schleef R.R. Loskutoff D.J. Blood. 1987; 70: 721-728Crossref PubMed Google Scholar). The nitrocellulose was blocked by incubation (1 h, 22°C) with 5% nonfat milk in PBS, 0.1% Tween 20. The washed blots were incubated (1 h, 22°C) with a primary antibody (i.e. monoclonal anti-decapeptide, 1 μg/ml (45Huse W.D. Sastry L. Iverson S.A. Kang A.S. Alting-Mees M. Burton D.R. Benkovic S.J. Lerner R.A. Science. 1989; 246: 1275-1281Crossref PubMed Scopus (685) Google Scholar); affinity-purified rabbit anti-A-61, 1 μg/ml, described below) diluted in phosphate-buffered saline (PBS; 50 mM phosphate, pH 7.4, 150 mM NaCl) supplemented with 0.1%" @default.
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