Di Marco S, Fendrich G, Knecht R, Strauss A, Pohlig G, Heim J, Priestle J P, Sommerhoff C P, Grütter M G
Core Drug Discovery Technologies, Pharmaceuticals Division, Ciba-Geigy Limited, Basel, Switzerland.
Protein Sci. 1997 Jan;6(1):109-18. doi: 10.1002/pro.5560060112.
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 alpha-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 A and 2.4 A, 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.
利用重叠寡核苷酸通过聚合酶链反应生成了编码55个氨基酸的蛋白质水蛭抑肽素的合成基因，该蛋白是来自医用水蛭的一种新型组织激肽释放酶抑制剂，将其与酵母α因子前导序列融合并在酿酒酵母中表达。重组水蛭抑肽素主要以未完全加工的融合蛋白形式分泌，但可使用酵母yscF蛋白酶的可溶性变体在体外进行加工。加工后的水蛭抑肽素纯化至纯度高于97%。N端序列分析和电喷雾电离质谱证实了N端加工正确以及预期的氨基酸序列和分子量。重组水蛭抑肽素的生物学活性与天然水蛭蛋白相同。单独结晶的水蛭抑肽素以及与组织激肽释放酶形成的复合物分别在1.4 Å和2.4 Å以上产生衍射。为了确定抑制剂的反应位点，通过监测溶液中的复合物形成以及抑制剂的蛋白水解切割来研究水蛭抑肽素与激肽释放酶、胰凝乳蛋白酶和胰蛋白酶的相互作用。在与高浓度、近乎等摩尔浓度的组织激肽释放酶孵育期间，水蛭抑肽素主要在假定的反应位点Arg 30和Ile 31之间的肽键处被切割，产生一种修饰的抑制剂。在与胰凝乳蛋白酶形成的相应复合物中，主要发现未切割的水蛭抑肽素，切割的水蛭抑肽素种类积累缓慢。与胰蛋白酶孵育导致水蛭抑肽素发生几次蛋白水解切割，主要的裂解肽键位于Arg 30和Ile 31之间。水蛭抑肽素似乎属于显示瞬时抑制作用的蛋白酶抑制剂类别。
