二硫键桥Cys^(2nd)-Cys^(6th)缺失对典型TIL类蛋白酶抑制剂抑制活性和特异性的影响  

Effect of disulfide bond Cys^(2nd)-Cys^(6th)disruption on the inhibitory activity and specificity of typical TIL protease inhibitors

作　　者：魏梦 王圆 张照锋 陈长清 李游山 WEI Meng;WANG Yuan;ZHANG Zhaofeng;CHEN Changqing;LI Youshan(College of Biological Science and Engineering,Shaanxi University of Technology,Hanzhong 723001,China;Qinba State Key Laboratory of Biological Resources and Ecological Environment(Incubation),Shaanxi University of Technology,Hanzhong 723001,China;Shaanxi Province Key Laboratory of Bio-resources,Shannxi University of Technology,Hanzhong 723001,China;Qinba Mountain Area Collaborative Innovation Center of Biological Comprehensive Development,Shaanxi University of Technology,Hanzhong 723001,China)

机构地区：[1]陕西理工大学生物科学与工程学院,陕西汉中723001 [2]陕西理工大学秦巴生物资源与生态环境省部共建国家重点实验室(培育),陕西汉中723001 [3]陕西理工大学陕西省资源生物重点实验室,陕西汉中7230012 [4]陕西理工大学陕南秦巴山区生物资源综合开发协同创新中心,陕西汉中723001

出　　处：《安徽大学学报（自然科学版）》2024年第1期85-96,共12页Journal of Anhui University(Natural Science Edition)

基　　金：国家自然科学基金青年科学基金资助项目(31702187);陕西省自然科学基础研究计划重点项目(2022JZ-12);陕西省教育厅重点科学研究计划项目(22JY017,20JY007);陕西理工大学科研项目(SLGKYXM2202);陕西高校青年创新团队(2023-77)。

摘　　要：笔者对AmCI,AsATI和AsC/E-1的编码序列进行密码子优化和基因合成,并利用点突变技术对其关键位点Cys^(2nd)和Cys^(6th)进行替换,结合原核表达和胶内活性染色分析,探讨Cys^(2nd)-Cys^(6th)对典型TIL类蛋白酶抑制剂抑制活性和抑制特异性的影响.SDS-PAGE结果显示,AmCI,AsATI和AsC/E-1及其突变体在上清中皆有表达.胶内活性染色结果显示,AmCI和AsC/E-1不仅能抑制胰凝乳蛋白酶和弹性蛋白酶活性,还能强烈抑制枯草杆菌蛋白酶活性,但对胰蛋白酶没有抑制作用.AsATI能抑制胰蛋白酶活性,却不能抑制胰凝乳蛋白酶、弹性蛋白酶和枯草杆菌蛋白酶活性.将Cys^(2nd)和Cys^(6th)分别替换为Asp和Leu后,AmCI,AsATI和AsC/E-1原有的抑制活性皆大大降低.值得注意的是,AsATI的突变体产生了微弱的枯草杆菌蛋白酶抑制活性.上述研究结果表明,Cys^(2nd)-Cys^(6th)对典型TIL类蛋白酶抑制剂的抑制活性和抑制特异性至关重要.笔者希望该研究不仅可为AmCI,AsATI和AsC/E-1的生理功能研究奠定基础,还能为TIL类蛋白酶抑制剂活性和特异性的定向改造提供一定的依据.The authors optimized the coding sequences of AmCI,AsATI,and AsC/E-1 through codon optimization and gene synthesis,and the key sites Cys^(2nd)and Cys^(6th)were replaced by site-specific mutagenesis.Combining prokaryotic expression and in-gel activity staining analysis,the influence of Cys^(2nd)-Cys^(6th)on inhibitory activity and specificity of typical TIL-type protease inhibitors was investigated.SDS-PAGE results showed that AmCI,AsATI,AsC/E-1 and their mutants were all expressed in the supernatant.The results of in-gel activity staining showed that AmCI and AsC/E-1 could not only inhibit the activities of chymotrypsin and elastase,but also strongly inhibit the activities of subtilisin,but had no inhibitory effect on trypsin.AsATI can inhibit trypsin activity,but cannot inhibit chymotrypsin,elastase and subtilisin activities.Cys^(2nd)and Cys^(6th)were replaced by Asp and Leu,respectively,the original inhibitory activities of AmCI,AsATI and AsC/E-1 were all greatly reduced.Notably,AsATI mutants produced weak inhibitory activity against subtilisin.The above results indicate that Cys^(2nd)-Cys^(6th)is crucial for the inhibitory activity and specificity of typical TIL protease inhibitors.The authors hoped that this study could not only lay the foundation for the physiological function study of AmCI,AsATI,and AsC/E-1,but also provide a certain basis for the directed modification of the activity and specificity of TIL-type protease inhibitors.

关 键 词：蛋白酶抑制剂 AmCI AsATI AsC/E-1 二硫键桥 抑制活性 抑制特异性 

分 类 号：Q553[生物学—生物化学]