Cloning, expression and purification of novel gene Rv2742 in Mycobacterium tuberculosis H37Rv / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 1771-1786, 2019.
Artículo
en Chino
| WPRIM
| ID: wpr-771754
ABSTRACT
Rv2742 is a novel gene identified from Mycobacterium tuberculosis H37Rv by the proteogenomics strategy. The aim of this study was to establish a system of soluble expression and purification of the missing protein Rv2742 in M. tuberculosis H37Rv, to provide reference for further research on the biological function of Rv2742. The soluble protein was not successfully induced by prokaryotic expression vectors pGEX-4T-2-Rv2742, pET-32a-Rv2742, pET-28a-Rv2742 and pMAL-c2X-Rv2742. After the codon of novel gene Rv2742 was optimized according to E. coli codon usage frequency, only the recombinant strain containing plasmid pMAL-c2X-Rv2742 could produce soluble products of Rv2742 encoding gene. In addition, the expression effects of the desired fusion protein were also analyzed under different conditions including hosts, culture temperatures and IPTG concentrations. The optimum expression conditions were as follows Rosetta (DE3) host, 16 °C culture temperature and 0.5 mmol/L IPTG. After being purified by affinity chromatography with amylose resin, the fusion protein sequence was confirmed by LC-MS/MS. These results indicated that the novel gene Rv2742 product could be successfully induced and expressed in a soluble form by the expression system pMAL-c2X with MBP tag. Our findings provide reference for studies on potential interaction and immunogenicity.
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Proteínas Recombinantes de Fusión
/
Cromatografía Liquida
/
Clonación Molecular
/
Escherichia coli
/
Espectrometría de Masas en Tándem
/
Genética
/
Mycobacterium tuberculosis
Tipo de estudio:
Estudio pronóstico
Idioma:
Chino
Revista:
Chinese Journal of Biotechnology
Año:
2019
Tipo del documento:
Artículo
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