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.

High production of bacteriorhodopsin from wild type Halobacterium salinarum

“…The optimum physical condition for maximum production of bR was a temperature of 39 °C, an agitation speed of 150 rpm, and light intensity of 6300 lux; under these conditions, productivity of bR improved from 0.26 to 1.11 mg/l h. The bR yield increased from 45.2 to 196 mg/l at 7 days, about 4.33-fold higher than in basal medium by adjusting the physical parameters and nitrogen sources. This is higher than the results reported by Kahaki et al (2014), who overproduced the bR gene in E. coli and obtained a bR concentration of 191 mg/l. Faezi Ghasemi et al (2008) increased the bR concentration about 3.49 fold over the use of basal medium by optimization of all parameters in culture medium; this was also lower than the results of the present study.…”

Discovery of bacteriorhodopsins in Haloarchaeal species isolated from Indian solar salterns: deciphering the role of the N‐terminal residues in protein folding and functional expression

“…Purification of BR was attempted using Ni-NTA chromatography, as described in the Experimental Procedures section, but no protein band corresponding to BR was observed after SDS-PAGE, perhaps due to the instability and protease sensitivity of recombinant BR (Cunningham and Deber, 2007). The Mistic tag has been shown to improve the expression of BR (Nekrasova et al, 2010;Kahaki et al, 2014). Four Mistic variants, M110, M2, M3 and M4, have been reported in the literature (Roosild et al, 2006;Dvir and Choe, 2009).…”

“…To address this issue, several N‐terminal exogenous tags have been used to help BR integrate into the bacterial inner membrane and increase the stability and yield to some extent. Examples include the use of carrier proteins such as a Mistic (Kahaki et al ., ), MBP (Chen and Gouaux, ), β‐lactamase (Karnik et al ., ; Thombre et al ., ) and ompA (Karnik et al ., ). However, the heterologous expression of BR using E. coli as an expression host has had limited success in reducing production costs partly due to the need for refolding in the presence of expensive detergents and lipids and/or the removal of recombinant fusion tags using expensive proteases.…”

Cloning and expression of phosphoenolpyruvate carboxykinase from a cestode parasite and its solubilization from inclusion bodies using l-arginine

“…Since none of host cells showed any improvement in rePEPCK solubility, E. coli BL21 was used to express the protein in the subsequent steps. The composition and richness of media have significant effect on protein expression and solubility [23]. rePEPCK construct was grown in different growth media; TB and 2xYT media resulted in greater cell mass than LB, however, they were not helpful in lowering the protein aggregation.…”

“…), and coexpression of molecular chaperones [20][21][22][23][24][25][26]. Heterologous proteins, expressed as IBs, can also be solubilized by adding solubilizing agents (guanidine hydrochloride (GdmCl), urea, N-lauroyl sarcosine, L-arginine, etc) [27,28].…”