摘要
目的确定重组泛素样特异性蛋白酶1(ubiquitin-like specific protease 1,Ulp1)在大肠杆菌中的最佳表达条件并进行分离纯化。方法采用DNA重组技术,构建重组质粒Ulp1-pET-28a,并转化到Escherichia coli BL21(DE3)细胞中,以IPTG诱导Ulp1原核表达,并从诱导时间、诱导温度和IPTG诱导浓度方面确定其最佳表达条件,再以亲和层析法分离纯化Ulp1。结果SDS-PAGE分析表明,工程菌诱导温度为30℃、诱导时间为6 h、IPTG浓度为0.2 mmol/L时Ulp1表达量最高,表达量约为350 mg/L且成功进行了Ulp1的分离纯化。结论本研究成功进行了Ulp1原核表达条件的优化与分离纯化,为Ulp1在生物工程中的酶切应用奠定了实验基础。
Objective To explore the optimal expression conditions of recombinant ubiquitin-like specific protease 1(Ulp1)in Escherichia coli and to isolate and purify it.Methods The recombinant plasmid Ulp1-pET-28a was constructed by using DNA recombinant technology and transformed into Escherichia coli BL21(DE3)cells to induce the prokaryotic expression of Ulp1 with IPTG.The optimal expression conditions was determined from induction time,induction temperature and IPTG concentration.Then Ulp1 was isolated and purified by affinity chromatography.Results SDS-PAGE analysis showed that the expression of Ulp1 was the highest,which was about 350 mg/L,when the recombinant cells were incubated for 6 hours at 30℃with 0.2 mmol/L IPTG,and Ulp1 was successfully isolated and purified.Conclusion In this study,the prokaryotic expression conditions of Ulp1 were successfully optimized,and Ulp1 was isolated and purified,laying an experimental foundation for the application of enzyme digestion of Ulp1 in bioengineering.
作者
张叶明
付正豪
陈云雨
Zhang Yeming;Fu Zhenghao;Chen Yunyu(Institute of Drug Screening and Evaluation,Wannan Medical College,Wuhu 241000 China)
出处
《锦州医科大学学报》
CAS
2020年第6期1-4,30,共5页
Journal of Jinzhou Medical University
基金
国家自然科学基金项目,项目编号:81703546
安徽省自然科学基金项目,项目编号:1808085QH265
吉林省科技发展计划项目,项目编号:20160520045JH
皖南医学院重点科研项目培育基金资助,项目编号:WK2018Z05。
关键词
泛素样特异性蛋白酶1
原核表达
表达优化
亲和层析
ubiquitin-like specific protease 1
prokaryotic expression
expression optimization
affinity chromatography
