Efficient Molecular Biological Manipulations with Improved Strategies Based on Novel Escherichia coli Vectors
Efficient Molecular Biological Manipulations with Improved Strategies Based on Novel Escherichia coli Vectors
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基于新型大肠杆菌载体的改进策略的高效分子生物操作
DOI：
10.1021/acs.jafc.1c00109
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发表时间：
2021
影响因子：
6.1
通讯作者：
An Yingfeng
中科院分区：
文献类型：
--
作者：
Zhang Yifeng;Gao Herui;Qi Xianghui;Gao Song;Xu Shumin;Wang Hongling;An Yingfeng
在本研究中，已经构建了一些新的质粒，灵活和零背景的分子克隆，更有效的表达，并与改进的策略纯化蛋白质。质粒pANY 4-pL 18-ccdB和pANY 4-pR 18/pL 18-ccdB在互补DNA链中具有不同的启动子。因此，异丙基-β-d-硫代半乳糖苷诱导或温度诱导的蛋白表达的重组质粒可以在单个分子克隆过程中同时构建，用于平行比较。有趣的是，当用于促进GFP或pfLamA酶的表达时，突变的pL 18和pR 18/pL 18启动子的表现与T7启动子相似或甚至更好。此外，构建了含有His-弹性蛋白样多肽（ELP）-内含肽多功能标签的质粒pANY 8，并设计了特殊的纯化方案以获得纯化的蛋白质，而不需要耗时的透析步骤来去除咪唑和高浓度的盐离子。此外，基于尿素的变性和重折叠过程可以方便地整合到ELP介导的沉淀方案中，用于纯化不溶性包涵体，省略了耗时的透析步骤。
In this study, some novel plasmids have been constructed for flexible and zero-background molecular cloning, more efficient expression, and purification of proteins with improved strategies. The plasmids pANY4-pL18-ccdB and pANY4-pR18/pL18-ccdB have different promoters in the complementary DNA strands. Therefore, recombinant plasmids for either isopropyl-β-d-thiogalactoside-induced or temperature-induced protein expression could be simultaneously constructed in a single molecular cloning process for parallel comparison. Intriguingly, the mutated pL18 and pR18/pL18 promoters performed similar to or even better than the T7 promoter when used for promoting the expression of the GFP or pfLamA enzyme. Moreover, the plasmid pANY8 containing the His-elastin-like polypeptide (ELP)-intein multifunctional tag was constructed, and special purification protocol was designed to obtain purified proteins without the requirement of time-consuming dialysis steps to remove imidazole and high concentration of salt ions. Additionally, the urea-based denaturation and refolding processes can be conveniently integrated into the ELP-mediated precipitation protocol for purification of insoluble inclusion bodies, omitting the time-consuming dialysis steps.