OVEREXPRESSION OF BACTERIOOPSIN IN ESCHERICHIA-COLI AS A WATER-SOLUBLE FUSION TO MALTOSE-BINDING PROTEIN - EFFICIENT REGENERATION OF THE FUSION PROTEIN AND SELECTIVE CLEAVAGE WITH TRYPSIN
Gq. Chen et Je. Gouaux, OVEREXPRESSION OF BACTERIOOPSIN IN ESCHERICHIA-COLI AS A WATER-SOLUBLE FUSION TO MALTOSE-BINDING PROTEIN - EFFICIENT REGENERATION OF THE FUSION PROTEIN AND SELECTIVE CLEAVAGE WITH TRYPSIN, Protein science, 5(3), 1996, pp. 456-467
Bacteriorhodopsin (bR) is a light-driven proton pump from Halobacteriu
m salinarium and is a model system for studying membrane protein foldi
ng, stability, function, and structure. bR is composed of bacterio-ops
in (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 purifi
cation epitope (1D4, nine amino acids) has been designed and synthesiz
ed. Overexpression of bO was achieved by fusion to the carboxyl termin
us of maltose binding protein (MBP). The expressed fusion protein (MBP
-sbO-1D4) formed inclusion bodies in Escherichia coli and, following s
olubilization with urea and removal of the urea by dialysis, approxima
tely 170 mg of similar to 75% pure MBP-sbO-1D4 was obtained from 1 L o
f culture. MBP-sbO-1D4 formed high molecular weight (greater than or e
qual to 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 pot
ential trypsin sites within bO, was accomplished by optimization of th
e digestion conditions. Both MBP-sbO-1D4 and sbO-1D4 were regenerated
with all-trans retinal and purified to homogeneity. In general, 6-10 m
g 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 absorbanc
e, light/dark adaptation, and photocycle properties were observed amon
g sbR-1D4, MBP-sbR-1D4, and bR from H. salinarium.