High-yield production of bacteriorhodopsin via expression of a synthetic gene in Escherichia coli.
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.
Pompejus
M
M
Institute für Molekulare Genetik, Georg-August-Universität Göttingen, Federal Republic of Germany.
Friedrich
K
K
Teufel
M
M
Fritz
H J
HJ
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005813:Genes, Synthetic; D005822:Genetic Vectors; D008823:Micelles; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011993:Recombinant Fusion Proteins
D008823:Micelles; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D001436:Bacteriorhodopsins