An efficient system for the synthesis of bacteriorhodopsin in Halobacterium halobium.
The mechanism by which bacteriorhodopsin (BR) transports protons across the cell membrane of Halobacterium halobium is actively studied in many laboratories. Currently available systems for the synthesis of mutant proteins obtained by site-directed mutagenesis of the gene encoding BR (bop) require reconstitution of the denatured polypeptide after its synthesis Escherichia coli or yeast; this approach is technically difficult and labor intensive, and raises questions about possible differences between in vivo and in vitro folding. Using a newly described transformation system and a halobacterial plasmid vector, we show that it is possible to reintroduce the bop gene into BR- strains of H. halobium. The bop-carrying plasmid expresses native BR in amounts similar to those obtained in several wild type strains. This system allows facile site-directed mutagenesis in halophilic archaebacteria.
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Wayne State University School of Medicine, Department of Biochemistry, Detroit, MI 48201.
Chang
M
M
Duschl
A
A
Lanyi
J
J
Needleman
R
R
D001436:Bacteriorhodopsins / Q000096:biosynthesis*; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006217:Halobacterium / Q000235:genetics*; D011994:Recombinant Proteins; D013057:Spectrum Analysis; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D001436:Bacteriorhodopsins