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Recombinant hirustasin: Production in yeast, crystallization, and interaction with serine proteases
Author(s) -
Marco Stefania Di,
Fendrich Gabriele,
Knecht Rene,
Strauss Andre,
Pohlig Gabriele,
Heim Jutta,
Priestle John P.,
Grütter Markus G.,
Sommerhoff Christian P.
Publication year - 1997
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560060112
Subject(s) - biochemistry , trypsin , kallikrein , proteases , peptide sequence , recombinant dna , saccharomyces cerevisiae , protease , microbiology and biotechnology , serine protease , biology , chymotrypsin , yeast , scissile bond , serine , chemistry , enzyme , gene
Abstract A synthetic gene coding for the 55‐amino acid protein hirustasin, a novel tissue kallikrein inhibitor from the leech Hirudo medicinalis , was generated by polymerase chain reaction using overlapping oligonucleotides, fused to the yeast α‐factor leader sequence and expressed in Saccharomyces 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 hirustasin was purified to better than 97% purity. N‐terminal sequence analysis and electrospray ionization mass spectrometry confirmed a correctly processed N‐terminus and the expected amino acid sequence and molecular mass. The biological activity of recombinant hirustasin was identical to that of the authentic leech protein. Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 Å and 2.4 Å, respectively. In order to define the reactive site of the inhibitor, the interaction of hirustasin with kallikrein, chymotrypsin, and trypsin was investigated by monitoring complex formation in solution as well as proteolytic cleavage of the inhibitor. During incubation with high, nearly equimolar concentration of tissue kallikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 31, the putative reactive site, to yield a modified inhibitor. In the corresponding complex with chymotrypsin, mainly uncleaved hirustasin was found and cleaved hirustasin species accumulated only slowly. 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 inhibitors displaying temporary inhibition.