Overexpression of bacterio-opsin in Escherichia coli as a water-soluble fusion to maltose binding protein: efficient regeneration of the fusion protein and selective cleavage with trypsin.
Bacteriorhodopsin (bR) is a light-driven proton pump from Halobacterium salinarium and is a model system for studying membrane protein folding, stability, function, and structure. bR is composed of bacterio-opsin (bO), the 248-amino acid apo protein, and all-trans retinal, which is linked to lysine 216 via a protonated Schiff base. A bO gene (sbOd) possessing 29 unique restriction sites and a carboxyl-terminal purification epitope (1D4, nine amino acids) has been designed and synthesized. Overexpression of bO was achieved by fusion to the carboxyl terminus of maltose binding protein (MBP). The expressed fusion protein (MBP-sbO-1D4) formed inclusion bodies in Escherichia coli and, following solubilization with urea and removal of the urea by dialysis, approximately 170 mg of approximately 75% pure MBP-sbO-1D4 was obtained from 1 L of culture. MBP-sbO-1D4 formed high molecular weight (> or = 2,000 kDa) oligomers that were water-soluble. The synthetic bO with the 1D4 tag (sbO-1D4) was separated from MBP by trypsin cleavage at the factor Xa site between the MBP and sbO-1D4 domains. Selective trypsin cleavage at the factor Xa site, instead of at the 14 other potential trypsin sites within bO, was accomplished by optimization of the digestion conditions. Both MBP-sbO-1D4 and sbO-1D4 were regenerated with all-trans retinal and purified to homogeneity. In general, 6-10 mg of sbR-1D4 and 52 mg of MBP-sbR-1D4 were obtained from 1 L of cell culture. No significant differences in terms of UV/vis light absorbance, light/dark adaptation, and photocycle properties were observed among sbR-1D4, MBP-sbR-1D4, and bR from H. salinarium.
Chen
G Q
GQ
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637, USA.
Gouaux
J E
JE
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001436:Bacteriorhodopsins / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015951:Factor Xa / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D008027:Light; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013053:Spectrophotometry; D014357:Trypsin / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D001436:Bacteriorhodopsins; C017827:bacterio-opsin; D014357:Trypsin; D015951:Factor Xa