High Overexpression and Purification of Optimized Bacterio-Opsin From Halobacterium Salinarum R1 in E. Coli Publisher Pubmed



Kahaki FA¹ ; Babaeipour V^{1, 4} ; Memari HR² ; Mofid MR³ Show Affiliations
Source: Applied Biochemistry and Biotechnology Published:2014

Abstract

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. © 2014, Springer Science+Business Media New York.