文献类型：期刊文献

中文题名：含非天然氨基酸定点突变的MLL3_(SET)蛋白表达与纯化

英文题名：Expression and Purification of the MLL3_(SET) Protein with a Site-directed Mutation of an Unnatural Amino Acid

作者：王小琴[1];黄银萍[1];王蔚倩[2];吴萍[2];全舒[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237;[2]中国科学院上海高等研究院国家蛋白质科学设施上海张江实验室,上海201210

年份：2022

卷号：38

期号：3

起止页码：194

中文期刊名：生物技术通报

外文期刊名：Biotechnology Bulletin

收录：CSTPCD;;北大核心:【北大核心2020】；CSCD:【CSCD2021_2022】；

基金：国家自然科学基金面上项目(31670802)。

语种：中文

中文关键词：MLL3_(SET);基因组改造;非天然氨基酸;表达纯化;酶活性

外文关键词：MLL3_(SET);genome modification;unnatural amino acid;expression and purification;enzyme activity

摘要：在组蛋白H3K4甲基转移酶MLL3的催化结构域(MLL3_(SET))中定点引入非天然氨基酸N-炔丙基赖氨酸(N-propargyl-lysine,PrK),表达、纯化该突变蛋白(MLL3_(SET)*),并评估突变蛋白的酶活,为后续进一步利用单分子荧光共振能量转移技术(smFRET)表征MLL3的作用机制奠定基础。将MLL3_(SET)接入pET-28b(+)构建表达载体,通过MLL3_(SET)晶体结构分析选择N4905位点进行PrK的引入;对商业化菌株(C321.ΔA.exp)进行基因组改造以引入T7 RNA聚合酶基因,并在改造后的菌株中表达、纯化MLL3_(SET)^(*),最后测定MLL3_(SET)^(*)的酶活性。结果表明,在构建的C321.ΔA.exp lacZ∷T7p07菌株中,pET28b-MLL3_(SET)^(*)在共转入aaRS-tRNA正交系统以及外源添加PrK后能够正常表达;通过Ni-NTA亲和层析及凝胶过滤层析成功纯化出高纯度的MLL3_(SET)^(*)蛋白;多酶级联反应结果显示,MLL3_(SET)^(*)的酶活性比野生型蛋白低,但仍具有约43%的甲基转移酶活性。本研究成功实现了含PrK的MLL3_(SET)蛋白的原核表达与纯化,突变蛋白保留了一定酶活性,为后续深入研究MLL3的分子机制奠定了基础。
This work aims to introduce the unnatural amino acid,N-propargyl-lysine(PrK),into the catalytic domain(MLL3_(SET))of histone H3K4 methyltransferase MLL3,to express and purify the mutant protein(MLL3_(SET)*),and evaluate its enzyme activity,which will lay the foundation for further single-molecule fluorescence resonance energy transfer(smFRET)experiments to characterize the mechanism of MLL3.MLL3_(SET) was linked into pET-28b(+)to construct an expression vector.Residue N4905 was selected for PrK introduction upon MLL3_(SET) crystal structure analysis.The commercial E.coli strain(C321.ΔA.exp)was genetically modified to integrate the T7 RNA polymerase gene.Further,MLL3_(SET)*was expressed and purified in the modified strain,and finally the enzyme activity was determined.The results showed that the constructed C321.ΔA.exp lacZ∷T7p07 strain harboring the pET28b-MLL3_(SET)*plasmid normally expressed the target protein after co-transformation with the aaRS-tRNA orthogonal system and the addition of PrK.The MLL3_(SET)*was purified successfully by Ni-NTA affinity chromatography and gel filtration chromatography.The results of the multi-component enzyme-coupled reaction showed that although the enzyme activity of MLL3_(SET)*was lower than that of wild-type MLL3_(SET),it retained about 43%of the wild-type enzyme activity.This study successfully achieves prokaryotic expression and purification of PrK labeled MLL3_(SET) protein retaining certain enzymatic activity,which lays a foundation for the subsequent in-depth study of the molecular mechanism of MLL3_(SET).