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

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

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tRNAIle 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，V_max为0.39 μmol/（L·min），K_m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。

Abstract:

[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 tRNAIle 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 V_max of 0.39 μmol/(L·min), K_m 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 beefficiently 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.