文献类型：期刊文献

中文题名：核酸去甲基酶AlkB在毕赤酵母中的表达纯化及在tRNA相关研究中的应用

英文题名：Nucleic acid demethylase AlkB in Pichia pastoris:expression,purification,and application in tRNA-related research

作者：崔璐瑶[1];李安娜[1];林茹[1];张嘉禛[1];施嘉诚[1];周勉[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

年份：2023

卷号：63

期号：3

起止页码：1088

中文期刊名：微生物学报

外文期刊名：Acta Microbiologica Sinica

收录：CSTPCD;;Scopus;北大核心:【北大核心2020】；CSCD:【CSCD2023_2024】；PubMed;

基金：国家重点研发计划(2018YFA0900300);上海市青年科技启明星计划(19QA1402600)。

语种：中文

中文关键词：毕赤酵母;去甲基酶;AlkB;tRNA;酶学性质

外文关键词：Pichia pastoris;demethylase;AlkB;tRNA;enzyme property

摘要：【目的】核酸的甲基化修饰是一种常见的化学修饰形式,具有重要的生物学功能,却也在一定程度上给一些核酸研究过程带来了技术难度。tRNA上具有的大量甲基化修饰会阻碍逆转录进程,从而降低荧光定量PCR(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)和高通量测序对其的检测效率。来自大肠杆菌(Escherichia coli)的AlkB蛋白是一种多功能的脱烷基化酶,可以去除DNA和RNA上多种甲基化为代表的修饰,有望解决以上问题。【方法】针对大肠杆菌来源的AlkB,分别尝试在大肠杆菌和毕赤酵母(Pichia pastoris)表达系统中进行诱导表达和纯化,对纯化获得的AlkB进行酶学性质测定。最后以tRNA^(Ile) _(UAU)等两种tRNA为代表,研究AlkB的处理对于荧光定量PCR法检测tRNA表达水平的影响。【结果】AlkB在大肠杆菌中表达时多以包涵体形式存在,但是在毕赤酵母中可以成功分泌表达。使用镍柱分离纯化后获得了纯度高于95%的AlkB蛋白,其酶学性质参数如下:最适反应温度为25℃,最适pH值为6.5,Vmax为0.39μmol/(L·min),Km为3.23μmol/L,比酶活为1.08 U/mg。AlkB对RNA的处理可以增加荧光定量PCR对tRNA表达水平检测的准确度。【结论】AlkB可以在毕赤酵母中高效表达并纯化,其处理有助于荧光定量PCR对于tRNA的检测结果变得更准确。AlkB的各项酶学性质特征在相关理论研究和应用中具有一定的科学价值。
[Objective]As a common type of chemical modification,nucleic acid methylation has significant biological functions.However,it also brings technical difficulties to some nucleic acid-related studies.Massive methylations on tRNAs will block reverse transcription and decrease the efficiency of real-time fluorescence quantitative PCR(RT-qPCR)and high-throughput sequencing for the determination of tRNA levels.The AlkB from Escherichia coli is a multi-functional dealkylase.It can remove methylation as well as other modifications on DNA and RNA and thus has the potential to solve the problem mentioned above.[Methods]Here we expressed the E.coli sourced AlkB in E.coli and Pichia pastoris.After purification of the protein,we measured its enzyme properties.Finally,two tRNAs represented by tRNA^(Ile)_(UAU) were used to examine the effect of AlkB treatment on the detection performance of real-time PCR for tRNA levels.[Results]AlkB mostly presented as inclusion bodies localized in E.coli,however,it was successfully expressed in and secreted by P.pastoris.After being purified by Nickel column,the AlkB protein showed the purity above 95%.The optimum conditions of this enzyme were 25℃and pH 6.5,at which it showed the Vmax of 0.39μmol/(L·min),Km of 3.23μmol/L,and specific activity of 1.08 U/mg.When RNA sample was treated by AlkB,real-time PCR could detect the tRNA level more accurately.[Couclusion]AlkB could be efficiently expressed and purified in P.pastoris.By treating RNA sample with purified AlkB,the real-time PCR method is able to detect tRNA levels more accurately.Additionally,the enzyme properties of AlkB have a significant value for relevant theoretical research and application.