BDELLASTASIN, A SERINE-PROTEASE INHIBITOR OF THE ANTISTASIN FAMILY FROM THE MEDICAL LEECH (HIRUDO-MEDICINALIS) - PRIMARY STRUCTURE, EXPRESSION IN YEAST, AND CHARACTERIZATION OF NATIVE AND RECOMBINANT INHIBITOR

We have reported earlier the isolation and amino acid composition of b dellin A from medical leech, and characterised it as an inhibitor of t rypsin, plasmin and acrosin [Fritz, H., Gebhardt, M., Meister, R. & Fi nk, E. (1971) in Proceedings of the international research conference on proteinase inhibitors (Fritz, H. & Tschesche, H., eds) pp. 271-280, Walter de Gruyter, Berlin]. In the present study one of several chrom atographic forms of this inhibitor was isolated from a semi-pure prepa ration. Elucidation of its amino acid sequence revealed that bdellin A is a member of the antistasin family. Therefore, it was renamed bdell astasin to avoid confusion with bdellin B, which is another trypsin-pl asmin inhibitor from the medical leech, but of the Kazal type. Further more, a synthetic gene of bdellastasin was constructed, and the protei n expressed in Saccharomyces cerevisiae with yields of 29 mg/l. The re combinant bdellastasin was purified by hydrophobic interaction and ani on-exchange chromatography. Comparison by mass spectroscopy, far-ultra violet circular dichroism studies, sequence determination, and inhibit ion characteristics demonstrated the identity of recombinant and nativ e bdellastasin. The K, values of bdellastasin for inhibition of bovine trypsin and human plasmin are in the nanomolar range; no inhibition w as detected for factor Xa, thrombin, tissue kallikrein, plasma kallikr ein and chymotrypsin. Circular dichroism analyses indicated that bdell astasin is devoid of secondary-structural elements.