Department of Biotechnology, Pukyong National University, Busan, Korea.
Appl Biochem Biotechnol. 2010 Nov;162(7):1858-71. doi: 10.1007/s12010-010-8964-6. Epub 2010 Apr 18.
Cathepsin L is an important protease in the initiation of protein degradation and one of the most powerful endopeptidases. In this study, we cloned mud loach (Misgurnus mizolepis) cathepsin L (MlCtL) cDNA, and the pro-mature enzyme of MlCtL (proMlCtL) was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in a pGEX-4 T-1 vector. The recombinant proMlCtL was overexpressed in E. coli DH5αMCR as a 62-kDa protein. Its activity was quantified by measuring the cleavage of synthetic fluorogenic peptide substrates, and the protease activity of proMlCtL was also demonstrated by gelatin zymography. Antipain and leupeptin were shown to inhibit the protease activity of proMlCtL. Our results suggest that the structural features and evolutionary relationship of the mud loach cathepsin L gene were similar to that of the other mammalian cathepsin Ls; however, the proMlCtL protein was more stable at neutral and alkaline pH. The optimum temperature for the proMlCtL enzyme was found to be 40 °C. In addition, proMlCtL activity was dependent upon the presence of several metal ions and detergents.
组织蛋白酶 L 是蛋白降解起始过程中的一种重要的蛋白酶，也是最强大的内肽酶之一。本研究克隆了泥鳅（Misgurnus mizolepis）组织蛋白酶 L（MlCtL）cDNA，并在 pGEX-4T-1 载体中，将 MlCtL 的前体酶（proMlCtL）表达为与谷胱甘肽 S-转移酶融合的蛋白。重组 proMlCtL 在大肠杆菌 DH5αMCR 中作为 62kDa 蛋白过表达。通过测量合成荧光肽底物的裂解来定量测定其活性，通过明胶酶谱法也证明了 proMlCtL 的蛋白酶活性。抗蛋白酶和亮抑酶肽被证明能抑制 proMlCtL 的蛋白酶活性。我们的结果表明，泥鳅组织蛋白酶 L 基因的结构特征和进化关系与其他哺乳动物的组织蛋白酶 Ls 相似；然而，proMlCtL 蛋白在中性和碱性 pH 值下更稳定。发现 proMlCtL 酶的最适温度为 40°C。此外，proMlCtL 的活性取决于几种金属离子和去污剂的存在。
