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Production of disulfide‐linked hirudin dimer by in vitro folding
Author(s) -
Chang Jui-Yoa,
Grossenbacher Hugo,
Meyhack Bernd,
Maerki Walter
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)81607-2
Subject(s) - hirudin , chemistry , dimer , monomer , cysteine , iodoacetamide , folding (dsp implementation) , biochemistry , thrombin , stereochemistry , organic chemistry , enzyme , biology , platelet , electrical engineering , immunology , engineering , polymer
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.