Isa Nuryana, Fina Amreta Laksmi, Eva Agustriana, Kartika Sari Dewi, Ade Andriani, Ahmad Thontowi, Wien Kusharyoto, Puspita Lisdiyanti
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引用次数: 1
Abstract
Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is the most frequently used enzyme in molecular biology for cDNA synthesis. To date, reverse transcription coupled with Polymerase Chain Reaction, known as RT-PCR, has been popular as an excellent approach for the detection of SARS-CoV-2 during the COVID-19 pandemic. In this study, we aimed to improve the enzymatic production and performance of MMLV-RT by optimizing both codon and culture conditions in E. coli expression system. By applying the optimized codon and culture conditions, the enzyme was successfully overexpressed and increased at high level based on the result of SDS-PAGE and Western blotting. The total amount of MMLV-RT has improved 85-fold from 0.002 g L−1 to 0.175 g L−1 of culture. One-step purification by nickel affinity chromatography has been performed to generate the purified enzyme for further analysis of qualitative and quantitative RT activity. Overall, our investigation provides useful strategies to enhance the recombinant enzyme of MMLV-RT in both production and performance. More importantly, the enzyme has shown promising activity to be used for RT-PCR assay.
Moloney小鼠白血病病毒逆转录酶(MMLV-RT)是分子生物学中最常用的cDNA合成酶。迄今为止,逆转录结合聚合酶链反应(RT-PCR)作为一种在COVID-19大流行期间检测SARS-CoV-2的极好方法而广受欢迎。在本研究中,我们旨在通过优化密码子和培养条件来提高MMLV-RT在大肠杆菌表达系统中的酶产率和性能。利用优化的密码子和培养条件,SDS-PAGE和Western blotting结果显示,该酶成功过表达并高水平表达。MMLV-RT的总量从0.002 g L−1提高到0.175 g L−1,提高了85倍。通过镍亲和层析一步纯化得到纯化酶,进一步进行RT活性的定性和定量分析。总之,我们的研究为提高MMLV-RT重组酶的产量和性能提供了有用的策略。更重要的是,该酶已显示出良好的活性,可用于RT-PCR检测。
期刊介绍:
The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.