Expression and enzymatic activity analysis of non-structure protein 14 of SARS-CoV-2
Objective To obtain the non-structure protein 14 (Nsp14) of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) with higher purity and enzymatic activity. Methods This study firstly analyzed the rare codons in the gene of nsp14 according to the codon usage bias of E. coli, followed by codon...
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| Format: | Article |
| Language: | zho |
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Institute of Basic Medical Sciences and Peking Union Medical College Hospital, Chinese Academy of Medical Sciences / Peking Union Medical College.
2023-01-01
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| Series: | Jichu yixue yu linchuang |
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| Online Access: | http://journal11.magtechjournal.com/Jwk_jcyxylc/fileup/1001-6325/PDF/1001-6325-2023-43-1-51.pdf |
| _version_ | 1827391065028558848 |
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| author | CHEN Shuang, XU Jingwen, DING Kaiyun, LIU Jiansheng, PENG Xiaozhong, LIU Hongqi |
| author_facet | CHEN Shuang, XU Jingwen, DING Kaiyun, LIU Jiansheng, PENG Xiaozhong, LIU Hongqi |
| author_sort | CHEN Shuang, XU Jingwen, DING Kaiyun, LIU Jiansheng, PENG Xiaozhong, LIU Hongqi |
| collection | DOAJ |
| description | Objective To obtain the non-structure protein 14 (Nsp14) of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) with higher purity and enzymatic activity. Methods This study firstly analyzed the rare codons in the gene of nsp14 according to the codon usage bias of E. coli, followed by codon optimization. The optimized nucleotide fragment of nsp14 was cloned into four kinds of expressing vectors respectively. Comparative analysis of yield and solubility was performed among these expressed four fusion proteins. The best one was chosen for further optimization of expressing conditions. After the fusion protein was purified by glutathione affinity column, the fusion tag was removed by 3C protease, and then the protein was purified by glutathione affinity column and molecular sieve column for further analysis of enzymatic activity through urea polyacrylamide gel electrophoresis. Results Many rare codons were found in expression of SARS-CoV-2 nsp14 in E. coli, among which some rare codons were distributed in close range and tandem. The best recombinant plasmid for expressing the fusion protein was pGEX6P1-GST-OPTI-Nsp14, which gave an eptimal expression in 30 ℃ with high yield and solubility. After purification, a higher purity of Nsp14 with nuclease activity was obtained. Conclusions This study shows that the SARS-CoV-2 Nsp14 protein with nuclease activity is successfully prepared, which lays a foundation for the further research on the structure and function of SARS-CoV-2 Nsp14, and provides favorable conditions for screening antiviral drugs targeting at Nsp14 of SARS-CoV-2. |
| first_indexed | 2024-03-08T17:03:18Z |
| format | Article |
| id | doaj.art-b039da4fb7144b748acdb4c70d9f707a |
| institution | Directory Open Access Journal |
| issn | 1001-6325 |
| language | zho |
| last_indexed | 2024-03-08T17:03:18Z |
| publishDate | 2023-01-01 |
| publisher | Institute of Basic Medical Sciences and Peking Union Medical College Hospital, Chinese Academy of Medical Sciences / Peking Union Medical College. |
| record_format | Article |
| series | Jichu yixue yu linchuang |
| spelling | doaj.art-b039da4fb7144b748acdb4c70d9f707a2024-01-04T07:27:26ZzhoInstitute of Basic Medical Sciences and Peking Union Medical College Hospital, Chinese Academy of Medical Sciences / Peking Union Medical College.Jichu yixue yu linchuang1001-63252023-01-01431515810.16352/j.issn.1001-6325.2023.01.0051Expression and enzymatic activity analysis of non-structure protein 14 of SARS-CoV-2CHEN Shuang, XU Jingwen, DING Kaiyun, LIU Jiansheng, PENG Xiaozhong, LIU Hongqi01. Department of Immunology, School of Basic Medicine of Kunming Medical University, Kunming 650500;;2. Institute of Medical Biology, CAMS & PUMC, Kunming 650118;;3. Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC, Beijing 100005, ChinaObjective To obtain the non-structure protein 14 (Nsp14) of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) with higher purity and enzymatic activity. Methods This study firstly analyzed the rare codons in the gene of nsp14 according to the codon usage bias of E. coli, followed by codon optimization. The optimized nucleotide fragment of nsp14 was cloned into four kinds of expressing vectors respectively. Comparative analysis of yield and solubility was performed among these expressed four fusion proteins. The best one was chosen for further optimization of expressing conditions. After the fusion protein was purified by glutathione affinity column, the fusion tag was removed by 3C protease, and then the protein was purified by glutathione affinity column and molecular sieve column for further analysis of enzymatic activity through urea polyacrylamide gel electrophoresis. Results Many rare codons were found in expression of SARS-CoV-2 nsp14 in E. coli, among which some rare codons were distributed in close range and tandem. The best recombinant plasmid for expressing the fusion protein was pGEX6P1-GST-OPTI-Nsp14, which gave an eptimal expression in 30 ℃ with high yield and solubility. After purification, a higher purity of Nsp14 with nuclease activity was obtained. Conclusions This study shows that the SARS-CoV-2 Nsp14 protein with nuclease activity is successfully prepared, which lays a foundation for the further research on the structure and function of SARS-CoV-2 Nsp14, and provides favorable conditions for screening antiviral drugs targeting at Nsp14 of SARS-CoV-2.http://journal11.magtechjournal.com/Jwk_jcyxylc/fileup/1001-6325/PDF/1001-6325-2023-43-1-51.pdfsevere acute respiratory syndrome coronavirus 2(sars-cov-2)|non-structure protein 14(nsp14)|prokaryotic expression|protein purification|nuclease activity |
| spellingShingle | CHEN Shuang, XU Jingwen, DING Kaiyun, LIU Jiansheng, PENG Xiaozhong, LIU Hongqi Expression and enzymatic activity analysis of non-structure protein 14 of SARS-CoV-2 Jichu yixue yu linchuang severe acute respiratory syndrome coronavirus 2(sars-cov-2)|non-structure protein 14(nsp14)|prokaryotic expression|protein purification|nuclease activity |
| title | Expression and enzymatic activity analysis of non-structure protein 14 of SARS-CoV-2 |
| title_full | Expression and enzymatic activity analysis of non-structure protein 14 of SARS-CoV-2 |
| title_fullStr | Expression and enzymatic activity analysis of non-structure protein 14 of SARS-CoV-2 |
| title_full_unstemmed | Expression and enzymatic activity analysis of non-structure protein 14 of SARS-CoV-2 |
| title_short | Expression and enzymatic activity analysis of non-structure protein 14 of SARS-CoV-2 |
| title_sort | expression and enzymatic activity analysis of non structure protein 14 of sars cov 2 |
| topic | severe acute respiratory syndrome coronavirus 2(sars-cov-2)|non-structure protein 14(nsp14)|prokaryotic expression|protein purification|nuclease activity |
| url | http://journal11.magtechjournal.com/Jwk_jcyxylc/fileup/1001-6325/PDF/1001-6325-2023-43-1-51.pdf |
| work_keys_str_mv | AT chenshuangxujingwendingkaiyunliujianshengpengxiaozhongliuhongqi expressionandenzymaticactivityanalysisofnonstructureprotein14ofsarscov2 |