Cloning, expression, and purification of recombinant protein from a single synthetic multivalent construct of Mycobacterium tuberculosis

• Published in: Protein expression and purification 2006-06, Vol.47 (2), p.341-347
• Main Authors: Fang, Chee-Mun, Zainuddin, Zainul F., Musa, Mustaffa, Thong, Kwai-Lin
• Format: Article
• Language: English
• Subjects: Antigens, Bacterial - biosynthesis; Antigens, Bacterial - genetics; Antigens, Bacterial - immunology; Antigens, Bacterial - isolation & purification; Cloning; Cloning, Molecular; Escherichia coli; Escherichia coli - genetics; Expression; Gene Expression; Inclusion body; Intranuclear Inclusion Bodies - immunology; Intranuclear Inclusion Bodies - metabolism; Multivalent; Mycobacterium tuberculosis; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - immunology; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - isolation & purification; Tuberculosis Vaccines - biosynthesis; Tuberculosis Vaccines - genetics; Tuberculosis Vaccines - immunology; Tuberculosis Vaccines - isolation & purification; Vaccines, Acellular - biosynthesis; Vaccines, Acellular - genetics; Vaccines, Acellular - immunology; Vaccines, Acellular - isolation & purification; VacIII
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2005.12.007

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 6× His-VacIII 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.