丝氨酸类蛋白酶对GST-Cry3Aa融合表达蛋白酶解活化分析  

Enzymatic Hydrolysis Analysis of the GST-Cry3Aa Fusion Protein by Serine Proteinase

作　　者：王亚芳[1] 翁士荷 徐卓吟 黄志成 肖霈 郭雅洁[2] 翁小倩[1] 吴松青[2] 

机构地区：[1]福建农林大学生命科学学院,福州350002 [2]福建农林大学林学院,福州350002

出　　处：《基因组学与应用生物学》2017年第12期5151-5158,共8页Genomics and Applied Biology

基　　金：福建省科技厅自然科学基金(No.2016J01097);国家自然科学基金(No.31601905);中央财政林业科技推广项目(No.K4315003A);福建农林大学科研基金(No.xjq201614);福建省大学生创新创业训练计划项目(No.201610389051;201610389055);国家大学生创新创业训练计划项目(No.201610389006)共同资助

摘　　要：苏云金芽胞杆菌(Bacillus thuringiensis,Bt)产生的Cry3Aa毒素在许多鞘翅目害虫的防治中发挥了重要作用。随着昆虫抗性的产生,如何通过大肠杆菌高通量表达筛选cry3Aa基因改造突变蛋白成为了研究的重点。然而,cry3Aa基因在大肠杆菌BL21(DE3)中诱导表达后其蛋白酶活化效果及杀虫活性均受到一定程度影响,对于影响的程度仍知之甚少。为探索cry3Aa基因所表达的融合蛋白GST-Cry3Aa中GST标签对Cry蛋白结构和功能的影响,本研究通过利用丝氨酸类蛋白酶(胰蛋白酶,糜蛋白酶)对纯化后的Cry3Aa融合蛋白进行水解条件研究分析,结果表明:在温度为11~37℃、GST-Cry3Aa与胰蛋白酶的质量比为4:1~30:1时,均可被水解出约55 k D的活性片段,而在同样条件下糜蛋白酶则无法对GST-Cry3Aa进行有效的水解活化。此外,结合I-TASSER和Swiss model对Cry3Aa与GST-Cry3Aa进行三维结构分析比对,发现GST-Cry3Aa在其结构域Ⅰ内有部分改变,推测其可能为两种丝氨酸类蛋白酶对该融合蛋白水解效果产生差异的原因。为后期构建Cry3Aa毒素高通量分子改造平台提供了理论基础。Cry3 Aa toxin, generated from Bacillus thuringiensis（Bt）, playes an important role in controlling of Coleoptera pests. With the pesticide resistant of insects produced, it was the focus of the study to use high throughput of Escherichia coli to express and screen modified cry3 Aa gene and protein. However, the toxin activation effect of protease and its pesticidal activity will be affected after the cry3 Aa gene was induced to express in Escherichia coli strain BL21（DE3） and little is known to th e impact. To explore the influence of GST-tag on the structure and function of Cry protein contained in GST-Cry3 Aa fusion protein, we used serine proteases（trypsin and chymotrypsin） to analyze the hydrolysis conditions for purified Cry3 Aa fusion protein. The results showed that under the condition of 11~37℃, with the mass ratio of GST-Cry3 Aa protein and trypsin from 4： 1 to 30： 1, trypsin cleavaged GST-Cry3 Aa protein into 55 k D activity segment while chymotrypsin couldn’t under the same condition. In addition, comparison analysis by I-TASSER and Swiss model for three-dimensional structure of Cry3 Aa protein and GST-Cry3 Aa protein demonstrated that there was the changes in domain I of GST-Cry3 Aa protein which might attribute to different hydrolysis effect of the two serine proteases providing the theoretical foundation for high throughput screening of modified Cry3 Aa protein.

关 键 词：Cry3Aa毒素 GST标签 丝氨酸类蛋白酶 酶解 

分 类 号：S476[农业科学—农业昆虫与害虫防治]