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High-yield production of bacteriorhodopsin via expression of a synthetic gene in Escherichia coli


High-yield production of bacteriorhodopsin via expression of a synthetic gene in Escherichia coli



European Journal of Biochemistry 211(1-2): 27-35



ISSN/ISBN: 0014-2956

PMID: 7916683

DOI: 10.1111/j.1432-1033.1993.tb19866.x

A gene (bos) coding for bacterioopsin (BO), the apoprotein of bacteriorhodopsin was assembled from chemically synthesized oligonucleotides by a new method of repeated rounds of insertion mutagenesis. The gene sequence was designed for convenient manipulation in future protein engineering experiments. In-frame fusion of bos to the lacZ454 gene allowed high-yield production in Escherichia coli of a beta-Gal454/BO fusion protein, deposited as intracellular inclusion bodies. These were enriched by virtue of their insolubility in 0.5% Triton X-100 and cleaved in aqueous suspension with IgA protease at a specific site provided at the beta-Gal454/BO boundary. Pure BO could be obtained from the mixture of water-insoluble cleavage products by selective extraction into organic solvent. The yield was in the range 30 - 50 mg pure protein/l culture medium, depending on individual preparation. This material could be used for reconstitution of fully functional bacteriorhodopsin. Taken together, the procedure constitutes a practical basis for the production of genetically engineered bacteriorhodopsins.

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Accession: 008775786

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