Production of disulfide-linked hirudin dimer by in vitro folding

• Published in: FEBS letters 1993-12, Vol.336 (1), p.53-56
• Main Authors: Chang, Jui-Yoa, Grossenbacher, Hugo, Meyhack, Bernd, Maerki, Walter
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biotechnology; BPTI, bovine pancreatic trypsin inhibitor; Cysteine - metabolism; Disulfide-linked hirudin dimerization; Disulfides - metabolism; Fundamental and applied biological sciences. Psychology; Health. Pharmaceutical industry; Hirudin dimer; Hirudins - genetics; Hirudins - metabolism; HV1, recombinant hirudin variant 1; Industrial applications and implications. Economical aspects; Kinetics; Other active biomolecules; Production of active biomolecules; Protein Folding; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - genetics
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(93)81607-2

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.