PRODUCTION OF DISULFIDE-LINKED HIRUDIN DIMER BY IN-VITRO FOLDING

A simple process of in vitro folding has been developed for the preparation of hirudin dimer. A variant of recombinant hirudin with Asp(33) replaced by Cys was expressed in yeast and isolated by HPLC. Crude Cys(33)-hirudin contains heterogeneous products that are made of one species of primary sequence. They were together reduced/denatured, and allowed to re-fold in the sodium bicarbonate buffer (pH 8.3) alone. Active, homogeneous Cys(33)-hirudin monomer folded spontaneously with a first order rate constant of 0.05 +/- 0.01 min(-1), followed by the oxidation of two Cys(33) to produce the pure dimer. The folding yield was 90%. On an equal weight basis, both Cys(33)-hirudin monomer and the dimer exhibit thrombin inhibitory activity comparable to that of wild-type hirudin. Due to the presence of an extra cysteine, the folding of active hirudin monomer (formation of three native disulfides) was accelerated by at least 12-fold.