Expression, purification, and activity assay of two new recombinant fibrinogen receptor antagonists

Aim: To construct and characterize two recombinant proteins, which are expected to be antagonists of fibrinogen receptors. Method: The gene sequence of decorsin which is extracted from a kind of North American leeches was synthesized. Two recombinant proteins, Annexin V plus decorsin (AnnV-D39) and Annexin V plus the carboxyl terminal 27 amino acids variant of decorsin (AnnV-D27), were constructed. And a 10 amino acids linker peptide of GGGGSGGGGS was inserted between annexin V and decorsin in AnnV-D39. Using pET-28 (a+) as an expressing vector, both two recombinant proteins were expressed in E. coli BL21 (DE3) with high efficiency as inclusion bodies. Result: The expression products were purified by DEAE-Cellulose 52 and Sepharose CL-4B chromatography under denaturing condition. The results of platelet aggregation assay (PAA) show that AnnV-D39 shows a good anti-platelet aggregation activity. However, AnnV-D27 shows no such activities in any PAA test. Conclusion: AnnV-D39 showed a good anti-platelet aggregation activity as a promising antagonist of fibrinogen receptor, while Annv-D27 needs further modification.