Abstract
A gene coding for the anticoagulant hirudin was synthesized and cloned into a yeast expression/secretion vector. The gene expression and secretion of hirudin was directed by the galactose-inducible promoter (GAL 10) and mating factor a pre-pro leader sequence. Biologically active hirudin was secreted into the extracellular medium. The initial expression level of hirudin gene in shake-flask culture was, however, very low (2.3 mg/litre). Modification of expression vector and optimization of fermentation conditions greatly improved the hirudin gene expression and secretion level into the culture supernatant more than 200 fold (460 mg/litre). Purification schemes for recombinant hirudin were also developed at a laboratory scale. Among different types of column chromatographies, immobilized metal affinity chromatography (IMAC) was found to be most efficient with respect to the purification fold and yield. Typical purification fold and yield obtained from a series of ion exchange, IMAC and gel filtration columns were 50 fold and 70%, respectively.