Expression, Purification and Activity Assay of Two New Recombinant Antagonists of Fibrinogen Receptor

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 acid residues 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. The expression products were purified by DEAE-Cellulose 52 and Sepharose CL-4B chromatography under denaturing condition. Platelet Aggregation Assay (PAA) shows that AnnV-D39 has good anti-platelet aggregation activity. However, AnnV-D27 shows no such activities in any PAA test. AnnV-D39 shows good anti-platelet aggregation activity as a new antagonist of fibrinogen receptor, while Annv-D27 needs re-modification.