Optimization of the expression system using galactose-inducible promoter for the production of anticoagulant hirudin in Saccharomyces cerevisiae.
We have tried to optimize the galactose-inducible gene expression system for the overproduction of the potent thrombin-specific inhibitor, hirudin, in a genetically engineered yeast, Saccharomyces cerevisiae. The expression and secretion of hirudin were directed by the galactose-inducible promoter, GAL10, and the mating factor alpha pre-pro leader sequence. The initial hirudin expression level in shake-flask culture was 2.3 mg 1-1. Modification of the expression vector and optimization of culture conditions, including the induction conditions, improved the level of hirudin gene expression and secretion into the culture supernatant more than 20-fold (50 mg 1-1) in a 4-1 scale batch cultivation. The expression and secretion level of hirudin seemed to be partially dependent on cell growth when galactose was used as a carbon source. Overexpression of the transcriptional activator, GAL4, appeared to have only negative effects on the expression of the hirudin gene and lacZ directed by the GAL10 promoter in the strain used in this study, unlike the previously reported examples. The complex medium containing yeast extract used for the increase of the cell mass and hirudin level did not show any detrimental effect on plasmid stability and did not complicate the downstream purification of hirudin from the culture supernatant. Moreover, the complex medium could greatly improve the hirudin productivity and reduce the degradation of hirudin produced in the culture supernatants.
Choi
E S
ES
Genetic Engineering Research Institute, KIST, Yusong, Taejon, Korea.
Sohn
J H
JH
Rhee
S K
SK
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D003470:Culture Media; D017931:DNA Primers / Q000235:genetics; D005690:Galactose / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors; D006629:Hirudins / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000187:drug effects; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D003470:Culture Media; D017931:DNA Primers; D006629:Hirudins; D005690:Galactose