Homologous overexpression of a light-driven anion pump in an archaebacterium.
The retinal protein halorhodopsin (HR), a light-driven chloride pump from Halobacterium halobium, was homologously overexpressed in this archaebacterium. Two DNA expression systems differing in their promoter region were investigated. The halopsin, hop, promoter coupled to the hop gene gave an increased level of HR synthesis. However, the extent of expression was driven by the copy number of the shuttle vector and did not reach the magnitude of the bacterio-opsin, bop, promoter system. Employing a gene fusion approach, the promoter for the bop gene was used to drive expression of the hop gene. A shuttle vector containing a bop-hop-cartridge was transformed into a HR-deficient strain and blueish-coloured transformants were obtained. The bop promoter expressed HR to an extent where a specific membrane fraction resembled the crystalline purple membrane of BR in terms of the lipid to protein ratio. HR could, therefore, be easily isolated in a natural membrane-bound state. This allows for direct use in biophysical studies without the application of detergents. This was the first successful overexpression of a 7-helical transmembrane protein and may be extended to other proteins of this family.
Max-Planck-Institut für Biochemie, Martinsried, Germany.
J A
JA
Heymann
W A
WA
Havelka
D
D
Oesterhelt
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002455:Cell Division; D004274:DNA, Recombinant; D005822:Genetic Vectors; D019617:Halobacterium salinarum / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D025602:Halorhodopsins; D016623:Ion Pumps / Q000502:physiology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D025602:Halorhodopsins; D016623:Ion Pumps; D011994:Recombinant Proteins; D001436:Bacteriorhodopsins; C017827:bacterio-opsin