Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae

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Abstract
Recombinant hirudin was produced by the yeast Saccharomyces cerevisiae using the .alpha.-pheromone prepro sequence to direct its secretion into the culture medium. The secreted hirudin was isolated to .gtoreq. 95% purity as measured by 205-nm absorbance integration from a reverse-phase chromatogram. One major activity peak corresponding to the complete, correctly processed molecule and two minor activity peaks corresponding to C-terminally truncated forms were identified. The primary structure of the major peak, determined by N-terminal sequencing of tryptic peptides, was that predicted from the cDNA sequence, and the molecular mass analyzed by fast atom bombardment mass spectrometry (FAB-MS) was 6892.6 (calculated 6892.5). UV spectral analysis suggested that, in contrast to the natural molecule, recombinant hirudin produced by S. cerevisiae is not sulfated.