Expression, Purification, and Characterization of Recombinant Ornatin E, a Potent Glycoprotein IIb-IIIa Antagonist

Abstract

A synthetic gene encoding ornatin E (OrnE), a 50-amino acid glycoprotein IIb-IIIa (GP IIb-IIIa) antagonist and platelet aggregation inhibitor isolated from the leech Placobdella ornata, was designed, constructed, and expressed in Escherichia coli. The OrnE gene was fused to the heat stable enterotoxin stil signal sequence and expressed under the transcriptional control of the E. coli alkaline phosphatase promoter. This construction directed secretion of recombinant ornatin E (rOrnE) into the extracellular medium at levels of 7-19 mg/liter. The protein was purified to apparent homogeneity in 18-38% yields by ammonium sulfate precipitation, Q-Sepharose and S-Sepharose ion exchange chromatography, and reverse-phase HPLC. Purified rOrnE was found to be indistinguishable from leech-derived OrnE as judged by amino acid composition, N-terminal sequencing, mass spectroscopic analysis, and HPLC coelution. In addition, rOrnE exhibits similar activity in fibrinogen/GP IIb-IIIa ELISA and platelet aggregation assays. Purified rOrnE possesses three disulfide bonds, the reduction and carboxymethylation of which results in a ca. 60-fold reduction in biological activity. A misfolded variant of rOrnE was characterized and shown to have a ca. 6-fold reduction in activity. These data demonstrate that the native disulfide bonds are required for the optimal GP IIb-IIIa antagonist activity of the ornatins.