Rapid high-yield purification and liposome reconstitution of polyhistidine-tagged sensory rhodopsin I.
We have used Ni(2+)-affinity chromatography as a rapid and efficient method to purify a sensory rhodopsin I (SR-I) derivative containing six consecutive 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 control of the bacterioopsin promoter. His-tagged SR-I retains native SR-I photochemical reactions in purified membranes and phototaxis signaling function in vivo. Immobilized Ni(2+)-affinity chromatography of membranes solubilized in 1% layryl maltoside provides a single-step purification of the protein to electrophoretic homogeneity (> or = 90% pure). The procedure yields 1.7 mg pure photoactive protein/liter of culture (60% efficiency). This yield combined with engineered overproduction of the protein provides at least 120-fold greater amounts than that of a previously reported multistep purification procedure, permitting structural and biochemical analysis previously not feasible. The purified protein in lauryl maltoside at pH 5.3 exhibits a visible absorption maximum at 587 nm characteristic of SR-I. Spectrometric titration reveals an alkaline-induced species at 550 nm previously observed with transducer-free SR-I in native membranes. A previously unreported structured absorption band at 400 nm, consistent with a deprotonated Schiff base, forms with the same pKa as the 550-nm species. His-tagged SR-I reconstituted into phosphatidylglycerol proteoliposomes retains properties of transducer-free SR-I in native membranes: its flash-induced absorption difference spectrum is identical, its photochemical reaction cycle kinetics show a similar pH dependence, and it forms a photoactive 550-nm species under alkaline conditions. These results indicate His-tagged SR-I reconstituted in proteoliposomes is suitable for analyzing SR-I interaction with its transducer protein in vitro.
Krebs
M P
MP
Department of Microbiology & Molecular Genetics, University of Texas-Medical School Health Science Center, Houston 77030, USA.
Spudich
E N
EN
Spudich
J L
JL
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D006217:Halobacterium / Q000737:chemistry / Q000235:genetics; D025602:Halorhodopsins; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010455:Peptides; D010777:Photochemistry; D011510:Proteolipids; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011984:Sensory Receptor Cells / Q000737:chemistry; D025603:Sensory Rhodopsins; D013053:Spectrophotometry
D017931:DNA Primers; D004269:DNA, Bacterial; D025602:Halorhodopsins; D010455:Peptides; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; C061497:SRI protein, Halobacterium; D025603:Sensory Rhodopsins; C020485:proteoliposomes; C033223:polyhistidine; D006639:Histidine; D001436:Bacteriorhodopsins