Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid.
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 alpha-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.
Shimono
Kazumi
K
RIKEN Systems and Structural Biology Center, Yokohama, Japan.
Goto
Mie
M
Kikukawa
Takashi
T
Miyauchi
Seiji
S
Shirouzu
Mikako
M
Kamo
Naoki
N
Yokoyama
Shigeyuki
S
D001436:Bacteriorhodopsins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D020355:Cholates / Q000737:chemistry; D002793:Cholic Acids / Q000737:chemistry; D004072:Digitonin / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry; D014176:Protein Biosynthesis
D020355:Cholates; D002793:Cholic Acids; D008081:Liposomes; D010713:Phosphatidylcholines; D001436:Bacteriorhodopsins; D004072:Digitonin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate