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

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.

Key words:Pichia pastoris;demethylase;AlkB;tRNA;enzyme property