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 Asp33 replaced by Cys was expressed in yeast and isolated by HPLC. Crude Cys33-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 Cys33-hirudin monomer folded spontaneously with a first order rate constant of 0.05 +/- 0.01 min-1, followed by the oxidation of two Cys33 to produce the pure dimer. The folding yield was 90%. On an equal weight basis, both Cys33-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.
Chang
J Y
JY
Pharmaceuticals Research Laboratories, Ciba-Geigy Ltd., Basel, Switzerland.
Grossenbacher
H
H
Meyhack
B
B
Maerki
W
W
D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D006629:Hirudins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004220:Disulfides; D006629:Hirudins; D011994:Recombinant Proteins; D003545:Cysteine