通过对东亚三角涡虫胰蛋白酶Djtry氨基酸序列比对分析, 发现保守的催化三联体结构中第一位的His被Lys所取代。为了探究这种突变是否会对胰蛋白酶的活性有影响, 文章构建了原核表达重组质粒pET-28a- Djtry, 转化到E.coli BL21中, 利用IPTG诱导表达, 对表达的重组蛋白进行变性、复性、纯化以及Western blotting鉴定, 获得成分均一的活性蛋白。利用牛胰蛋白酶为标准品, 胰蛋白酶特异性底物BAEE, 检测Djtry酶活力与比活力。SDS-PAGE电泳表明诱导表达的融合蛋白为包涵体, 分子量约为26 kDa, Western blotting结果显示为目的蛋白, 对复性纯化的目的蛋白进行酶活检测发现, 突变型胰蛋白酶Djtry仍然保持了胰蛋白酶催化性质, 但是催化活性相对较弱。
The cDNA Djtry, encoding a planarian trypsin, was identified from the cDNA library of Dugesia japonica. Multiple alignment analysis showed that the Tryps_SPc domain contained the incompletely conserved catalytic triad in which the first amino acid His was substituted by Lys. Phylogenetic analysis indicateed that Djtry protein falls at the base of other animal trypsins. The Djtry cDNA was cloned into a bacterial vector pET-28a and was transferred into E. coli BL21. The His-tagged Djtry fusion protein expression was induced by IPTG. SDS-PAGE analysis revealed that the Djtry was expressed as inclusion bodies in E. coli BL21 with the estimated molecular weight of approximately 26 kDa. Western blotting with His-tag antibody showed that the antibody was reacted with the fusion protein after refolding. Compared to bovine trypsin using BAEE as special substrate of trypsin, the enzyme activity of Djtry was measured. These results indicate that Djtry represents the archetype of animal trypsins, and this type of mutational trypsin Djtry still performs the trypsin nature with slightly weaker activity.
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