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

Abstract

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 β‐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 β‐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/1 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. Copyright © 1993, Wiley Blackwell. All rights reserved