RAPID HIGH-YIELD PURIFICATION AND LIPOSOME RECONSTITUTION OF POLYHISTIDINE-TAGGED SENSORY RHODOPSIN-1

We have used Ni2+-affinity chromatography as a rapid and efficient met hod to purify a sensory rhodopsin I (SR-I) derivative containing six c onsecutive histidine residues at its C-terminus (His-tagged SR-I), The protein was expressed in Halobacterium salinarium by integrating the corresponding gene at the chromosomal bacterioopsin locus under the co ntrol of the bacterioopsin promoter, His-tagged SR-I retains native SR -I photochemical reactions in purified membranes and phototaxis signal ing function in vivo. Immobilized Ni2+-affinity chromatography of memb ranes solubilized in 1% lauryl maltoside provides a single-step purifi cation of the protein to electrophoretic homogeneity (greater than or equal to 90% pure). The procedure yields 1.7 mg pure photoactive prote in/liter of culture (60% efficiency). This yield combined with enginee red overproduction of the protein provides at least 120-fold greater a mounts than that of a previously reported multistep purification proce dure, permitting structural and biochemical analysis previously not fe asible. The purified protein in lauryl maltoside at pH 5.3 exhibits a visible absorption maximum at 587 nm characteristic of SR-I. Spectrome tric titration reveals an alkaline-induced species at 550 nm previousl y observed with transducer-free SR-I in native membranes. A previously unreported structured absorption band at 400 nm, consistent with a de protonated Schiff base, forms with the same pK(a) as the 550-nm specie s. His-tagged SR-I reconstituted into phosphatidylglycerol proteolipos omes retains properties of transducer-free SR-I in native membranes: i ts flash-induced absorption difference spectrum is identical, its phot ochemical reaction cycle kinetics show a similar pH dependence, and it forms a photoactive 550-nm species under alkaline conditions. These r esults indicate His tagged SR-I reconstituted in proteoliposomes is su itable for analyzing SR-I interaction with its transducer protein in v itro. (C) 1995 Academic Press, Inc.