Hirudin, a protein found in the salivary gland of the medicinal leech (Hirudo medicinalis), is the most potent thrombin inhibitor known. Thrombin is a crucial component of the blood clotting cascade. The high specificity of thrombin inhibition renders Hirudin an excellent anticoagulant for therapeutic and diagnostic purposes. The recombinant Hirudin is produced in strains of the methylotrophic yeast Hansenula polymorpha. The expression is controlled by a strong, stringently regulated promoter derived from a gene of the methanol metabolism pathway. The degradation at the C-terminus of the polypeptide is not pronounced in this species as encountered in other yeast strains. A comprehensive analysis of the purified product confirms its proper folding and processing. A process for the production of recombinant Hirudin derived from the yeast expression system Hansenula polymorpha has been established. The production strain is characterized by high mitotic stability of the multi-copy expression cassettes, resulting in highly reproducible fermentation yields. The non-glycosylated and non-sulfated, biologically active protein (Desulfato-Hirudin) is secreted to the culture medium at levels of up to several g/L.
