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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

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【作者】 王军唐建国张庭芳张龙翔

【Author】 Wang Jun; Tang Jian-Guo; Zhang Ting-Fang; Zhang Long-Xiang(College of Life Sciences, Peking University, Beijing 100871)

【机构】 北京大学生命科学学院

【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。

【Abstract】 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.

【基金】 国家863计划基金
  • 【分类号】Q556.9
  • 【被引频次】16
  • 【下载频次】253
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