RECOMBINANT HIRUSTASIN - PRODUCTION IN YEAST, CRYSTALLIZATION, AND INTERACTION WITH SERINE PROTEASES

A synthetic gene coding for the 55-amino acid protein hirustasin, a no vel tissue kallikrein inhibitor from the leech Hirudo medicinalis, was generated by polymerase chain reaction using overlapping oligonucleot ides, fused to the yeast ct-factor leader sequence and expressed in Sa ccharomyces cerevisiae. Recombinant hirustasin was secreted mainly as incompletely processed fusion protein, but could be processed in vitro using a soluble variant of the yeast yscF protease. The processed hir ustasin was purified to better than 97% purity. N-terminal sequence an alysis and electrospray ionization mass spectrometry confirmed a corre ctly processed N-terminus and the expected amino acid sequence and mol ecular mass. The biological activity of recombinant hirustasin was ide ntical to that of the authentic leech protein. Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 Ang strom and 2.4 Angstrom, respectively In order to define the reactive s ite of the inhibitor, the interaction of hirustasin with kallikrein, c hymotrypsin, and trypsin was investigated by monitoring complex format ion in solution as well as proteolytic cleavage of the inhibitor. Duri ng incubation with high, nearly equimolar concentration of tissue kall ikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 31, the putative reactive site, to yield a modified inhibit or. In the corresponding complex with chymotrypsin, mainly uncleaved h irustasin was found and cleaved hirustasin species accumulated only sl owly. Incubation with trypsin led to several proteolytic cleavages in hirustasin with the primary scissile peptide bond located between Arg 30 and Ile 31. Hirustasin appears to fall into the class of protease i nhibitors displaying temporary inhibition.