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Structure-function studies on bacteriorhodopsin. I. Expression of the bacterio-opsin gene in Escherichia coli

Structure-function studies on bacteriorhodopsin. I. Expression of the bacterio-opsin gene in Escherichia coli

Journal of Biological Chemistry 262(19): 9246-9254

ISSN/ISBN: 0021-9258

PMID: 3298252

To express the bacterio-opsin (bop) gene in Escherichia coli, we have employed the inducible expression vectors pIN-II-A, -B, and -C (Nakamura, K., and Inouye, M. (1982) EMBO J. 1, 771-775). The vectors contain three cloning sites early in the E. coli lipoprotein gene (lpp) which is transcribed from tandem lpp and lac promoters. The bop gene was modified so as to delete the N-terminal leader sequence and then cloned into each of the three cloning sites to encode three different lipoprotein/bacterio-opsin fusions. Expression of the fusions was demonstrated both in vitro and in vivo. The fusion protein was estimated to be about 0.05% of the total cell protein. The cause for the low level of expression apparently was neither an inadequate level of mRNA nor degradation of the protein. However, expression of the fusions caused inhibition of the growth of the host to varying extents. One fusion protein was purified from E. coli membranes to homogeneity by immunoaffinity chromatography followed by preparative gel electrophoresis. The purified fusion protein generated a bacteriorhodopsin-like chromophore on treatment with defined lipid/detergent mixtures and retinal. When reconstituted into vesicles, the protein pumped protons on illumination comparably to the reconstituted native bacterio-opsin.

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