Cloning, Expression, and Purification of Recombinant Protein from a Single Synthetic Multivalent Construct of Mycobacterium Tuberculosis
Tuberculosis remains a major infectious disease with over 8 million new cases and 2 million deaths annually. Therefore, a vaccine more potent than BCG is desperately needed. In this regard, an approximately 800 bp DNA encoding a mycobacterial synthetic gene designated as VacIII (containing ubiquitin...
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Elsevier
2006
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| author | Fang, Chee Mun Zainuddin, Zainul F. Musa, Mustaffa Thong, Kwai Lin |
| author_facet | Fang, Chee Mun Zainuddin, Zainul F. Musa, Mustaffa Thong, Kwai Lin |
| author_sort | Fang, Chee Mun |
| collection | UM |
| description | Tuberculosis remains a major infectious disease with over 8 million new cases and 2 million deaths annually. Therefore, a vaccine more potent than BCG is desperately needed. In this regard, an approximately 800 bp DNA encoding a mycobacterial synthetic gene designated as VacIII (containing ubiquitin gene UbGR and four immunogenic mycobacterial epitopes or genes of ESAT-6, Phos1, Hsp 16.3, and Mtb8.4) was sub-cloned into a bacterial expression vector of pRSET-B resulting in a 6x His-VaeIII fusion gene construction. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8 M urea and the recombinant protein was purified by Ni-NTA column and dialyzed by urea gradient dialysis. This method produced a relatively high yield of recombinant VacIII protein and the cloned VacIII gene offers the potential development of other vaccine formats such as DNA vaccine and recombinant vaccine. (c) 2006 Elsevier Inc. All rights reserved. |
| first_indexed | 2024-03-06T05:14:31Z |
| format | Article |
| id | um.eprints-5485 |
| institution | Universiti Malaya |
| last_indexed | 2024-03-06T05:14:31Z |
| publishDate | 2006 |
| publisher | Elsevier |
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| spelling | um.eprints-54852018-10-15T03:19:39Z http://eprints.um.edu.my/5485/ Cloning, Expression, and Purification of Recombinant Protein from a Single Synthetic Multivalent Construct of Mycobacterium Tuberculosis Fang, Chee Mun Zainuddin, Zainul F. Musa, Mustaffa Thong, Kwai Lin Q Science (General) QH Natural history QR Microbiology Tuberculosis remains a major infectious disease with over 8 million new cases and 2 million deaths annually. Therefore, a vaccine more potent than BCG is desperately needed. In this regard, an approximately 800 bp DNA encoding a mycobacterial synthetic gene designated as VacIII (containing ubiquitin gene UbGR and four immunogenic mycobacterial epitopes or genes of ESAT-6, Phos1, Hsp 16.3, and Mtb8.4) was sub-cloned into a bacterial expression vector of pRSET-B resulting in a 6x His-VaeIII fusion gene construction. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8 M urea and the recombinant protein was purified by Ni-NTA column and dialyzed by urea gradient dialysis. This method produced a relatively high yield of recombinant VacIII protein and the cloned VacIII gene offers the potential development of other vaccine formats such as DNA vaccine and recombinant vaccine. (c) 2006 Elsevier Inc. All rights reserved. Elsevier 2006 Article PeerReviewed Fang, Chee Mun and Zainuddin, Zainul F. and Musa, Mustaffa and Thong, Kwai Lin (2006) Cloning, Expression, and Purification of Recombinant Protein from a Single Synthetic Multivalent Construct of Mycobacterium Tuberculosis. Protein Expression and Purification, 47 (2). pp. 341-347. ISSN 1046-5928, DOI https://doi.org/10.1016/j.pep.2005.12.007 <https://doi.org/10.1016/j.pep.2005.12.007>. https://doi.org/10.1016/j.pep.2005.12.007 doi:10.1016/j.pep.2005.12.007 |
| spellingShingle | Q Science (General) QH Natural history QR Microbiology Fang, Chee Mun Zainuddin, Zainul F. Musa, Mustaffa Thong, Kwai Lin Cloning, Expression, and Purification of Recombinant Protein from a Single Synthetic Multivalent Construct of Mycobacterium Tuberculosis |
| title | Cloning, Expression, and Purification of Recombinant Protein from a Single Synthetic Multivalent Construct of Mycobacterium Tuberculosis |
| title_full | Cloning, Expression, and Purification of Recombinant Protein from a Single Synthetic Multivalent Construct of Mycobacterium Tuberculosis |
| title_fullStr | Cloning, Expression, and Purification of Recombinant Protein from a Single Synthetic Multivalent Construct of Mycobacterium Tuberculosis |
| title_full_unstemmed | Cloning, Expression, and Purification of Recombinant Protein from a Single Synthetic Multivalent Construct of Mycobacterium Tuberculosis |
| title_short | Cloning, Expression, and Purification of Recombinant Protein from a Single Synthetic Multivalent Construct of Mycobacterium Tuberculosis |
| title_sort | cloning expression and purification of recombinant protein from a single synthetic multivalent construct of mycobacterium tuberculosis |
| topic | Q Science (General) QH Natural history QR Microbiology |
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