High Overexpression and Purification of Optimized Bacterio-Opsin from Halobacterium Salinarum R1 in E. coli

• Published in: Applied biochemistry and biotechnology Vol. 174; no. 4; pp. 1558 - 1571
• Main Authors: Kahaki, Fatemeh Abarghooi, Babaeipour, Valiollah, Memari, Hamid Rajabi, Mofid, Mohammad Reza
• Format: Journal Article
• Language: English
• Published: New York Springer-Verlag 01.10.2014; Springer US; Springer; Springer Nature B.V
• Subjects: Bacillus subtilis; Bacillus subtilis - genetics; Bacteriology; Bacteriorhodopsins - biosynthesis; Bacteriorhodopsins - chemistry; Bacteriorhodopsins - genetics; Bacteriorhodopsins - isolation & purification; Biochemistry; Biological and medical sciences; Biotechnology; Chemistry; Chemistry and Materials Science; E coli; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; genes; Halobacterium; Halobacterium salinarum; Halobacterium salinarum - genetics; membrane proteins; Membranes; Proteins; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; temperature; Bacterio-opsin; Membrane protein; Endogenous expression; Mistic; Purification; Archaeobacteria; Gene overexpression; Optimization; Protein; Halobacterium salinarum; Halobacteriales; Bacteria; Membrane; Opsin; Halobacteriaceae
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-014-1137-2

The purple membrane of Halobacterium Salinarum carries out a protein, bacteriorhodopsin (bR), which is a model for structure–function studies of membrane proteins. The heterologous expression of integral membrane proteins (IMPS) is difficult. In this study, we reported the heterologous overexpression of bacterio-opsin (bO) in Escherichia coli BL21 (DE3). Bacterio-opsin expression is facilitated by using mistic, a membrane protein from Bacillus subtilis in E. coli BL21 (DE3) membranes. The optimized bO gene was cloned in fusion to the C-terminus of mistic in pET 30a (+) and contains an oct-histidine in C-terminal to facilitate purification. Different medium, temperature, and induction time were used to optimize protein overexpression. The highest expression was obtained from the Terrific Broth (TB) medium at 18 °C with an IPTG concentration of 0.1 mM. The final purified bR was 192 ± 1 mg/L which has an important value for the production of membrane proteins in E. coli.