title: High overexpression and purification of optimized bacterio-opsin from Halobacterium Salinarum R1 in E. coli.
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.
authors.affiliation: Department of Life Science Engineering, Faculty of New Technologies, University of Tehran, Tehran, Iran.
authors.forename: Fatemeh Abarghooi
authors.initials: FA
authors.lastname: Kahaki
authors.affiliation: Department of Life Science Engineering, Faculty of New Technologies, University of Tehran, Tehran, Iran. vbabai@gmail.com.|Department of Biological Science and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran. vbabai@gmail.com.
authors.forename: Valiollah
authors.initials: V
authors.lastname: Babaeipour
authors.affiliation: Center of Biotechnology and Life Sciences and School of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
authors.forename: Hamid Rajabi
authors.initials: HR
authors.lastname: Memari
authors.affiliation: Department of Biochemistry and Bioinformatics Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Mofid
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics; D001436:Bacteriorhodopsins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D019617:Halobacterium salinarum / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011993:Recombinant Fusion Proteins; D001436:Bacteriorhodopsins