Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

Abstract

Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane a-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.

Structural Mechanism for Light-driven Transport by a New Type of Chloride Ion Pump, Nonlabens marinus Rhodopsin-3

“…Overall Structure and Comparison with Those of Bacteriorhodopsin, Halorhodopsins, and KR2-We synthesized the fulllength NM-R3 protein, using an E. coli cell-free protein production system (23,24). We obtained over 25 mg of NM-R3 from the 9-ml reaction mixture.…”

“…Cell-free Protein Synthesis and Purification-Cell-free protein synthesis of NM-R3 was performed essentially according to the previously reported protocols used for Acetabularia rhodopsin I production (22)(23)(24). Briefly, the cell-free reaction mixture included 100 M all-trans-retinal, 0.4% digitonin, and 6.67 mg/ml egg yolk phosphatidylcholine.…”

Allosteric regulation of γ-secretase activity by a phenylimidazole-type γ-secretase modulator

“…3217-v; Peptide institute) (21). Recombinant γ-secretase complex by Sf9 cells and T4 lysozyme-human PS1 chimeric protein by an E. coli cell-free protein synthesis system were prepared as described previously (19,22). Full descriptions of experiments are detailed in SI Appendix, SI Materials and Methods.…”

“…These results are consistent with the results of the photoaffinity labeling experiment, demonstrating that ST1120 facilitates the formation of the proteolytically active conformation of the catalytic center of γ-secretase. Furthermore, we tested the effect of ST1120 on the intrinsic proteolytic activity of the recombinant PS protein produced by an Escherichia coli-based cellfree protein synthesis system (22) (Fig. 2C).…”

Crystal Structure of the Eukaryotic Light-Driven Proton-Pumping Rhodopsin, Acetabularia Rhodopsin II, from Marine Alga

“…The latter was named ARII. The cell-free protein synthesis system supplemented with a steroid-derived detergent and lipid 28 successfully generated a large amount of ARII, which allowed us to perform various experiments. We analyzed the retinal configuration and the photochemistry.…”