Vu Thi Duy Ly, T. T. Giang, Ngo Thu Trang, Dinh Nho Thai, Phan Tuấn Nghĩa, Nguyen Thi Hong Loan
{"title":"Cloning and Expression of 3 Chymotrypsin-like Protease of SARS-CoV-2 in E. coli using pET28a Vector","authors":"Vu Thi Duy Ly, T. T. Giang, Ngo Thu Trang, Dinh Nho Thai, Phan Tuấn Nghĩa, Nguyen Thi Hong Loan","doi":"10.25073/2588-1140/vnunst.5509","DOIUrl":null,"url":null,"abstract":"The 3 chymotrypsin-like protease (3CLpro) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is one of the primary targets for the development of antiviral drug therapies as it plays a critical role in viral replication. In this study, the gene encoding for SARS-CoV-2 3CLpro (918 bp) was amplified from the cDNA of the virus by polymerase chain reaction (PCR) and cloned into the pGEM-T vector. 3CLpro was then inserted into the expression vector pET28a at the end of the 6 histidine residue encoding sequence to form a fusion protein (6xHis-3CLpro). The 6xHis-3Clpro construct was successfully expressed in E. coli. The expression of 3CLpro was highest when E. coli BL21(DE3) RIL harboring pET28a-3CLpro vector was cultured in LB medium at 20 oC, induced by 1.0 mM Isopropyl thiogalactopyranosie (IPTG) when cell density measured by optical density at 600 nm (OD600) reached 0.7-0.8 and harvested after 24 hours of induction. The recombinant 3CLpro was purified by Ni-sepharose affinity chromatography under denaturation conditions. The purified 3CLpro showed to have a 41 kDa band on sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using polyclonal anti-3CLpro antibody and hydrolyzed a fluorescent specific substrate of 3CLpro after renaturation.","PeriodicalId":23524,"journal":{"name":"VNU Journal of Science: Natural Sciences and Technology","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2023-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"VNU Journal of Science: Natural Sciences and Technology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.25073/2588-1140/vnunst.5509","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 1
Abstract
The 3 chymotrypsin-like protease (3CLpro) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is one of the primary targets for the development of antiviral drug therapies as it plays a critical role in viral replication. In this study, the gene encoding for SARS-CoV-2 3CLpro (918 bp) was amplified from the cDNA of the virus by polymerase chain reaction (PCR) and cloned into the pGEM-T vector. 3CLpro was then inserted into the expression vector pET28a at the end of the 6 histidine residue encoding sequence to form a fusion protein (6xHis-3CLpro). The 6xHis-3Clpro construct was successfully expressed in E. coli. The expression of 3CLpro was highest when E. coli BL21(DE3) RIL harboring pET28a-3CLpro vector was cultured in LB medium at 20 oC, induced by 1.0 mM Isopropyl thiogalactopyranosie (IPTG) when cell density measured by optical density at 600 nm (OD600) reached 0.7-0.8 and harvested after 24 hours of induction. The recombinant 3CLpro was purified by Ni-sepharose affinity chromatography under denaturation conditions. The purified 3CLpro showed to have a 41 kDa band on sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using polyclonal anti-3CLpro antibody and hydrolyzed a fluorescent specific substrate of 3CLpro after renaturation.