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.