Smith S M, Gottesman M M
Howard Hughes Medical Institute, National Cancer Institute, Bethesda, Maryland 20892.
J Biol Chem. 1989 Dec 5;264(34):20487-95.
A cDNA clone encoding the human cysteine protease cathepsin L was expressed at high levels in Escherichia coli in a T7 expression system. The insoluble recombinant enzyme was solubilized in urea and refolded at alkaline pH. 38-kDa procathepsin L was purified by gel filtration at pH 8.0, and a 29-kDa form of the enzyme was purified by gel filtration after autoprocessing of the proenzyme at pH 6.5. The kinetic properties of the 29-kDa species of recombinant cathepsin L were similar to those published for the human liver enzyme (Mason, R. W., Green, G. D. J., and Barrett, A.J. (1985) Biochem. J. 226, 233-241), using benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide as substrate. However, the stability of the recombinant enzyme, and its pH optimum for this substrate was shifted to a higher pH. Structure-function studies of cathepsin L were performed by constructing mutations in either the propeptide portion or the carboxyl-terminal light chain portion of the protein. These constructions were expressed in the E. coli system, and enzymatic activities were assayed following solubilization, renaturation, and gel filtration chromatography of the mutated proteins. Deletions of increasing size in the propeptide resulted in large proportional losses of activity, indicating that the propeptide is essential for proper enzyme folding and/or processing in this renaturation system. Deletion of part of the light chain containing a disulfide-forming cysteine residue or a single amino acid substitution of alanine for this cysteine residue resulted in almost complete loss of activity. These data suggest that the disulfide bond joining the heavy and light chains of cathepsin L is essential for enzymatic activity.
一个编码人半胱氨酸蛋白酶组织蛋白酶L的cDNA克隆在T7表达系统中于大肠杆菌中高水平表达。不溶性重组酶在尿素中溶解并在碱性pH下复性。38 kDa的组织蛋白酶L原通过在pH 8.0下的凝胶过滤进行纯化，而该酶的29 kDa形式在pH 6.5下对酶原进行自加工后通过凝胶过滤进行纯化。以苄氧羰基-苯丙氨酸-精氨酸-7-(4-甲基)香豆素酰胺为底物时，重组组织蛋白酶L的29 kDa形式的动力学性质与已发表的人肝脏酶的动力学性质相似（梅森，R.W.，格林，G.D.J.，和巴雷特，A.J.（1985年）《生物化学杂志》226，233 - 241）。然而，重组酶的稳定性及其对该底物的最适pH向更高的pH偏移。通过在蛋白质的前肽部分或羧基末端轻链部分构建突变来进行组织蛋白酶L的结构 - 功能研究。这些构建体在大肠杆菌系统中表达，并且在对突变蛋白进行溶解、复性和凝胶过滤色谱分析后测定酶活性。前肽中大小不断增加的缺失导致活性大幅成比例丧失，表明前肽对于该复性系统中酶的正确折叠和/或加工至关重要。缺失包含形成二硫键的半胱氨酸残基的部分轻链或用丙氨酸对该半胱氨酸残基进行单个氨基酸取代导致活性几乎完全丧失。这些数据表明连接组织蛋白酶L重链和轻链的二硫键对于酶活性至关重要。
