Archae-opsin expressed in Escherichia coli and its conversion to purple pigment in vitro.
Archae-opsin-1 (aO-1) has been expressed efficiently as a fusion protein with 13 heterologous amino acids at the amino terminus of the mature aO-1 in Escherichia coli under the control of T7 promoter. The E. coli-expressed aO-1, designated as aO-1002, which was located in the membrane fraction, was extracted with 8 M urea and partially purified by gel filtration chromatography in the presence of SDS. When all-trans retinal was added, aO-1002 in dimyristoylphosphatidylcholine and detergent-mixed micelles was converted to a purple pigment with lambda max at 558 nm at 20 degrees C via a 435/460 nm intermediate. Conversion of the intermediate to purple pigment was the rate-limiting step and proceeded as a two-state transition, because an isosbestic point was seen at 485 nm. Similar spectral changes were also observed in the regeneration process of hydroxylamine-bleached claret membranes and aO-1 isolated from claret membranes. Thus, the polypeptide of aO-1002 is considered to fold and form a retinal binding pocket in phospholipid and detergent micelles similarly to aO-1 isolated from the halobacterial membranes. Purple pigment showed a light-driven proton-pumping activity when reconstituted into phosphatidylcholine liposomes.
Sugiyama
Y
Y
Department of Biology, Faculty of Science, Nagoya University, Aichi.
Mukohata
Y
Y
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001426:Bacterial Proteins / Q000502:physiology*; D001483:Base Sequence; D004926:Escherichia coli; D005822:Genetic Vectors; D008027:Light; D008969:Molecular Sequence Data; D010860:Pigments, Biological / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017494:Proton Pumps; D011994:Recombinant Proteins / Q000096:biosynthesis; D012172:Retinaldehyde / Q000378:metabolism; D017299:Rod Opsins / Q000096:biosynthesis / Q000235:genetics*
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D010860:Pigments, Biological; D017494:Proton Pumps; D011994:Recombinant Proteins; D017299:Rod Opsins; C090129:archae-opsin 1 protein, Halobacterium; C055476:archaerhodopsin protein, Archaea; D012172:Retinaldehyde