PURIFICATION OF STORAGE GRANULE PROTEIN-23 - A NOVEL PROTEIN IDENTIFIED BY PHAGE DISPLAY TECHNOLOGY AND INTERACTION WITH TYPE-1 PLASMINOGEN-ACTIVATOR INHIBITOR

Type 1 plasminogen activator inhibitor (PAI-1) is a key regulator of t he fibrinolytic cascade that is stored in a rapidly releasable form wi thin platelet alpha-granules. To identify proteins that may participat e in the targeting or storage of this potent inhibitor, this report in vestigates the applicability of utilizing filamentous bacteriophages t o display proteins expressed by cells containing a regulated secretory pathway and their enrichment based upon an interaction with PAI-1, Fo r this purpose, RNA was extracted from AtT-20 cells (i.e. a classical model cell system for intracellular protein sorting), reverse transcri bed, amplified using polymerase chain reaction primers containing inte rnal restriction sites, and cloned into the phagemid pCOMB3H for expre ssion as fusion constructs with the bacteriophage gene III protein, Es cherichia coli was transformed with the phagemids and infected with VC SM13 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 vect or system that replaces the gene III protein with a decapeptide tag fo r immunologic quantitation, One novel cDNA clone (i.e. A-61), which pr eferentially recognized solution-phase PAI-1 and reacted positively wi th antibodies derived from a rabbit immunized with alpha-granules, was subcloned into the prokaryotic expression vector pTrcHis to create a construct containing an N-terminal six-histidine purification tag, Thi s construct was expressed in E. coli, purified by nickel-chelate chrom atography followed by preparative SDS-polyacrylamide gel electrophores is, and utilized for the generation of polyclonal antibodies, Immunobl otting analysis employing antibodies against the purified A-61 constru ct revealed a 23-kDa protein present in the regulated secretory pathwa y of AtT-20 cells, The 23-kDa molecule was purified from media conditi oned 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-Sepharo se, N-terminal amino acid sequencing of a 16-kDa Lys-C proteolytic fra gment of the 23-kDa storage granule protein was employed to confirm it s identity with the cDNA sequence of clone A-61, These data indicate t hat phage display of cDNA libraries fused to the C-terminal region of the gene III protein and their enrichment via an interaction with a ta rget molecule can be utilized to define other proteins present within a particular cellular pathway.