PROCESS-DEVELOPMENT FOR THE PRODUCTION OF RECOMBINANT HIRUDIN IN SACCHAROMYCES-CEREVISIAE - FROM UPSTREAM TO DOWNSTREAM

A gene coding for the anticoagulant hirudin was synthesized and cloned into a yeast expression/secretion vector. The gene expression and sec retion of hirudin was directed by the galactose-inducible promoter (GA L10) and mating factor a pre-pro leader sequence. Biologically) active hirudin was secreted into the extracellular medium. The initial expre ssion 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 expressio n 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 chro matographies, immobilized metal affinity chromatography (IMAC) was fou nd to be most efficient with respect to the purification fold and yiel d. Typical purification fold and yield obtained from a series of ion e xchange, IMAC and gel filtration columns were 50 fold and 70%, respect ively.