胰蛋白酶分子中二硫键Cys129－Cys232的定位改造研究  

Studies on the Site-directed Mutagenesis of the Disulfide Linkage Cys129-Cys232 of Trypsin

作　　者：王军[1] 唐建国[1] 张庭芳[1] 张龙翔[1] 

机构地区：[1]北京大学生命科学学院

出　　处：《生物化学杂志》1996年第5期583-587,共5页

基　　金：国家863计划基金

摘　　要：对胰蛋白酶所特有的二硫键［１２９，２３２］进行了定位改造，将Ｃｙｓ突变为Ｓｅｒ，以观察其对胰蛋白酶稳定性及活性的影响。采用蛋白质工程的方法，构建了三个突变体Ｃ１２９Ｓ、Ｃ２３２Ｓ和Ｃ１２９Ｓ／Ｃ２３２Ｓ．在Ｅ．ｃｏｌｉＸ－９０菌体中进行表达，表达产物用含胰蛋白酶特异性底物ＴＡＭＥ的活性胶检测活性，发现Ｃ２３２Ｓ丧失胰蛋白酶活性，而Ｃ１２９Ｓ和Ｃ１２９Ｓ／Ｃ２３２Ｓ保留了胰蛋白酶活性。在盐酸胍作用下比较双突变体和野生型胰蛋白酶活性，发现突变体Ｃ１２９Ｓ／Ｃ２３２Ｓ的稳定性有所降低。结果表明二硫键Ｃｙｓ１２９－Ｃｙｓ２３２对于胰蛋白酶的活性是非必需的，可能在稳定蛋白质的结构上发挥着重要作用。Two oligodeoxynucleotide were synthesized and used as primers for site-directed mutagenesis to replace either one or both cysteine residues of the disulfide linkage Cys129-Cys232 of trypsin. Clones from transformed E. coli JM101 were picked and screened by DNA sequencing. to facilitate the detection of trypsin activity, plasmids PT3-C232S, PT-C129S and pT3-C129S/ C2325 were constructed and heterologously expressed in E. coli X-90 which was used as the host. Periplasmic proteins including trypsin secreted were fractoinated by CM-Sepharose Fast Flow and the activity of trypsin was assayed by active polyacrylamide gel impregnated with TAME and phenol red. the apperance of yellow spots on a purple background indicated trypsin activity. Every time the mutant trypsin C129S and C129S/C2325 appeared as yellow spots, but never did C232S. It indicated the mutant C129S and the double mutant C129S/C232S retained their activity while the mutant C232S lost its activity. Denaturation experiment with guanidine hydrochloride showed that the stability of the double mutant C129S/C232S was lower as compared with-type trypsin. Quantitative experiment on kinetics and stability properties are in progress.

关 键 词：胰蛋白酶 二硫键 定位突变 

分 类 号：Q556.9[生物学—生物化学]