High‐yield production of bacteriorhodopsin via expression of a synthetic gene in Escherichia coli

Abstract

A gene (bos) coding for bacterioopsin (BO), the apoprotein of bacteriorhodopsin was assembled from chemically synthesized oligonucleotides by a new method of repeated rounds of insertion mutagenesis. The gene sequence was designed for convenient manipulation in future protein engineering experiments. In-frame fusion of bos to the lacZ454 gene allowed high-yield production in Escherichia coli of a fl-Ga1454/BO fusion protein, deposited as intracellular inclusion bodies. These were enriched by virtue of their insolubility in 0.5% Triton X-100 and cleaved in aqueous suspension with IgA protease at a specific site provided at the P-Ga1454/BO boundary. Pure BO could be obtained from the mixture of water-insoluble cleavage products by selective extraction into organic solvent. The yield was in the range 30-50 mg pure protein/l culture medium, depending on individual preparation. This material could be used for reconstitution of fully functional bacteriorhodopsin. Taken together, the procedure constitutes a practical basis for the production of genetically engineered bacteriorhodopsins.

Expression of membrane proteins from Mycobacterium tuberculosis in Escherichia coli as fusions with maltose binding protein

“…However, relatively few transmembrane proteins and interfacial membrane proteins have been tested for expression as fusions with the large MBP [1,14,15,29,30]. Fusions of hydrophobic membrane proteins with the hydrophilic MBP domain have been shown to reduce toxicity and improve stability for bacterio-opsin and bacteriorodopsin [35,36]. This study reports on the expression of multiple membrane proteins using MPB as a fusion partner.…”

A new hybrid protein for production of recombinant bacteriorhodopsin in Escherichia coli

“…1c). A similar stage of protein digestion described for ␤-Gal454/bO and MBP-bO fusions (Chen and Gouaux, 1996;Pompejus et al, 1993) resulted in at least twofold loss of bO. Thus, high efficiency of proteolysis in our work largely contributed to the overall productivity of the procedure for bO purification.…”

“…To purify bO and to obtain a functionally active recombinant bR, we followed a common approach, which involves extraction of bO from insoluble fraction with organic solvents or detergents, subsequent transfer of the target protein to SDS solution, and, finally, renaturation of bR in lipid-detergent micelles in the presence of retinal (Braiman et al, 1987;Miercke et al, 1991;Pompejus et al, 1993;Chen and Gouaux, 1996). As shown in Fig.…”

“…The highest level of renatured bR expressed in this way was 17 mg/l Miercke et al, 1991), however, uncontrolled proteolysis of the target protein in the cell resulted in non-homogenous protein preparation. Later a hybrid protein approach was developed, which favoured overexpression of bO in the form of inclusion bodies (Chen and Gouaux, 1996;Pompejus et al, 1993) and, thus, alleviated problems connected with target protein instability and toxicity to cells. Maltose-binding protein (MBP) and a large fragment of ␤-galactozidase (454 aa) were successfully used as N-terminal carrier proteins, and expression levels of about 200 mg/l were achieved for these protein fusions.…”

Short Communication

“…Before measuring light-driven proton translocation (right panel) the samples were dialyzed for two times 6 h in the dark against 50 volumes of 1 % (w/v) octyl glucoside in water at 0 °C. Proteoliposomes were prepared using soy bean lipids and proton translocation experiments were performed as described by Pompejus et al (1993). Arrows indicate time points of switching light on or off or addition of HOI, as indicated.…”