A novel method for synthesizing authentic SARS-CoV-2 main protease

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-06-08 DOI:10.1016/j.pep.2024.106531

Cheng Zhao, Yi Rong, Shuyuan Shi, Wen-chao Gao, Chaofeng Zhang

{"title":"A novel method for synthesizing authentic SARS-CoV-2 main protease","authors":"Cheng Zhao, Yi Rong, Shuyuan Shi, Wen-chao Gao, Chaofeng Zhang","doi":"10.1016/j.pep.2024.106531","DOIUrl":null,"url":null,"abstract":"<div>The SARS-CoV-2 main protease (Mpro) plays a crucial role in virus amplification and is an ideal target for antiviral drugs. Currently, authentic Mpro is prepared through two rounds of proteolytic cleavage. In this method, Mpro carries a self-cleavage site at the N-terminus and a protease cleavage site followed by an affinity tag at the C-terminus. This article proposes a novel method for producing authentic Mpro through single digestion. Mpro was constructed by fusing a His tag containing TEV protease cleavage sites at the N-terminus. The expressed recombinant protein was digested by TEV protease, and the generated protein had a decreased molecular weight and significantly increased activity, which was consistent with that of authentic Mpro generated by the previous method. These findings indicated that authentic Mpro was successfully obtained. Moreover, the substrate specificity of Mpro was investigated. Mpro had a strong preference for Phe at position the P2, which suggested that the S2 subsite was an outstanding target for designing inhibitors. This article also provides a reference for the preparation of Mpro for sudden coronavirus infection in the future.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106531"},"PeriodicalIF":1.4000,"publicationDate":"2024-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein expression and purification","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1046592824001037","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}

引用次数: 0

Abstract

The SARS-CoV-2 main protease (M^pro) plays a crucial role in virus amplification and is an ideal target for antiviral drugs. Currently, authentic M^pro is prepared through two rounds of proteolytic cleavage. In this method, M^pro carries a self-cleavage site at the N-terminus and a protease cleavage site followed by an affinity tag at the C-terminus. This article proposes a novel method for producing authentic M^pro through single digestion. M^pro was constructed by fusing a His tag containing TEV protease cleavage sites at the N-terminus. The expressed recombinant protein was digested by TEV protease, and the generated protein had a decreased molecular weight and significantly increased activity, which was consistent with that of authentic M^pro generated by the previous method. These findings indicated that authentic M^pro was successfully obtained. Moreover, the substrate specificity of M^pro was investigated. M^pro had a strong preference for Phe at position the P2, which suggested that the S2 subsite was an outstanding target for designing inhibitors. This article also provides a reference for the preparation of M^pro for sudden coronavirus infection in the future.

查看原文本刊更多论文

合成正宗 SARS-CoV-2 主要蛋白酶的新方法。

SARS-CoV-2 主要蛋白酶（Mpro）在病毒扩增过程中起着至关重要的作用，是抗病毒药物的理想靶标。目前，正宗的 Mpro 是通过两轮蛋白酶裂解制备的。在这种方法中，Mpro 的 N 端带有自裂解位点，C 端带有蛋白酶裂解位点和亲和标签。本文提出了一种通过单一消化生产真品 Mpro 的新方法。通过在 N 端融合含有 TEV 蛋白酶裂解位点的 His 标签，构建了 Mpro。表达的重组蛋白经 TEV 蛋白酶消化后，生成的蛋白分子量降低，活性显著提高，与之前方法生成的正宗 Mpro 蛋白一致。这些结果表明成功获得了正宗的 Mpro。此外，还对 Mpro 的底物特异性进行了研究。Mpro 对 P2 位上的 Phe 有强烈的偏好，这表明 S2 位是设计抑制剂的绝佳靶点。本文还为今后制备冠状病毒突变感染的 Mpro 提供了参考。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.