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Modification of SARS-CoV S1 gene render expression in Pichia pastoris
S1 gene fragment containing receptor-binding region was amplified by several sets of primers using Over-Lap PCR. The native S1 gene was modified at A + T abundant regions; n.t.777–1683, n.t.1041–1050, n.t.1236–1248, n.t.1317–1335, n.t.1590–1605; based on the same amino acid sequences. The modified g...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Kluwer Academic Publishers
2006
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7088608/ https://www.ncbi.nlm.nih.gov/pubmed/16991004 http://dx.doi.org/10.1007/s11262-006-0072-x |
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author | Lu, Haisong Yang, Guoliang Fei, Xiaozhan Guo, Hongyan Tan, Yadi Chen, Huanchun Guo, Aizhen |
author_facet | Lu, Haisong Yang, Guoliang Fei, Xiaozhan Guo, Hongyan Tan, Yadi Chen, Huanchun Guo, Aizhen |
author_sort | Lu, Haisong |
collection | PubMed |
description | S1 gene fragment containing receptor-binding region was amplified by several sets of primers using Over-Lap PCR. The native S1 gene was modified at A + T abundant regions; n.t.777–1683, n.t.1041–1050, n.t.1236–1248, n.t.1317–1335, n.t.1590–1605; based on the same amino acid sequences. The modified gene was cloned into a yeast expression vector pPIC9K. The resultant plasmid pPIC9K- S1 was transformed into Pichia pastoris GS 115 and the protein expression was induced with methanol. SDS-PAGE confirmed that the recombinant SI was secreted in the supernatant of induced GS 115. The protein yield reached 69 mg/l. ELISA and Western blot demonstrated that the S1 could react with the convalescent sera of people infected by SARS-CoV. Furthermore, ligand blot assay showed that the recombinant S1 could react with ACE2, the receptor of SARS-CoV. The molecular mass of expressed S1 was about 70 kDa, which was higher than that of the 30 kDa expected. PNGase F deglycosylation resulted in a protein band of 30 kDa. In conclusion, the S1 gene modification rendered the high-level expression of S1 in P. pastoris GS 115 and the protein was secreted as a biologically active form which was hyperglycosylated. |
format | Online Article Text |
id | pubmed-7088608 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2006 |
publisher | Kluwer Academic Publishers |
record_format | MEDLINE/PubMed |
spelling | pubmed-70886082020-03-23 Modification of SARS-CoV S1 gene render expression in Pichia pastoris Lu, Haisong Yang, Guoliang Fei, Xiaozhan Guo, Hongyan Tan, Yadi Chen, Huanchun Guo, Aizhen Virus Genes Original Article S1 gene fragment containing receptor-binding region was amplified by several sets of primers using Over-Lap PCR. The native S1 gene was modified at A + T abundant regions; n.t.777–1683, n.t.1041–1050, n.t.1236–1248, n.t.1317–1335, n.t.1590–1605; based on the same amino acid sequences. The modified gene was cloned into a yeast expression vector pPIC9K. The resultant plasmid pPIC9K- S1 was transformed into Pichia pastoris GS 115 and the protein expression was induced with methanol. SDS-PAGE confirmed that the recombinant SI was secreted in the supernatant of induced GS 115. The protein yield reached 69 mg/l. ELISA and Western blot demonstrated that the S1 could react with the convalescent sera of people infected by SARS-CoV. Furthermore, ligand blot assay showed that the recombinant S1 could react with ACE2, the receptor of SARS-CoV. The molecular mass of expressed S1 was about 70 kDa, which was higher than that of the 30 kDa expected. PNGase F deglycosylation resulted in a protein band of 30 kDa. In conclusion, the S1 gene modification rendered the high-level expression of S1 in P. pastoris GS 115 and the protein was secreted as a biologically active form which was hyperglycosylated. Kluwer Academic Publishers 2006 /pmc/articles/PMC7088608/ /pubmed/16991004 http://dx.doi.org/10.1007/s11262-006-0072-x Text en © Springer Science+Business Media, LLC 2006 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. |
spellingShingle | Original Article Lu, Haisong Yang, Guoliang Fei, Xiaozhan Guo, Hongyan Tan, Yadi Chen, Huanchun Guo, Aizhen Modification of SARS-CoV S1 gene render expression in Pichia pastoris |
title | Modification of SARS-CoV S1 gene render expression in Pichia pastoris |
title_full | Modification of SARS-CoV S1 gene render expression in Pichia pastoris |
title_fullStr | Modification of SARS-CoV S1 gene render expression in Pichia pastoris |
title_full_unstemmed | Modification of SARS-CoV S1 gene render expression in Pichia pastoris |
title_short | Modification of SARS-CoV S1 gene render expression in Pichia pastoris |
title_sort | modification of sars-cov s1 gene render expression in pichia pastoris |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7088608/ https://www.ncbi.nlm.nih.gov/pubmed/16991004 http://dx.doi.org/10.1007/s11262-006-0072-x |
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