Purification of storage granule protein-23. A novel protein identified by phage display technology and interaction with type I plasminogen activator inhibitor.
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 alpha-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 alpha-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.
Lang
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Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA. rschleef@riscsm.scripps.edu
Chuang
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TL
Barbas
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Schleef
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D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D017395:Plasminogen Activator Inhibitor 1; D011506:Proteins; C102602:storage granule protein 23