TY - JOUR
T1 - Expression of salinarum halorhodopsin in Escherichia coli cells
T2 - Solubilization in the presence of retinal yields the natural state
AU - Yamashita, Yasutaka
AU - Kikukawa, Takashi
AU - Tsukamoto, Takashi
AU - Kamiya, Masakatsu
AU - Aizawa, Tomoyasu
AU - Kawano, Keiichi
AU - Miyauchi, Seiji
AU - Kamo, Naoki
AU - Demura, Makoto
N1 - Funding Information:
We thank Prof. John L. Spudich, at the University of Texas-Houston Medical School, for providing the pJS010 plasmid and Pho81Wr - strain. This study was supported by a grant from the Japanese Ministry of Education, Culture, Sports, Science, and Technology to T.K. ( 23510251 ).
PY - 2011/12
Y1 - 2011/12
N2 - Salinarum halorhodopsin (HsHR), a light-driven chloride ion pump of haloarchaeon Halobacterium salinarum, was heterologously expressed in Escherichia coli. The expressed HsHR had no color in the E. coli membrane, but turned purple after solubilization in the presence of all-trans retinal. This colored HsHR was purified by Ni-chelate chromatography in a yield of 3-4 mg per liter culture. The purified HsHR showed a distinct chloride pumping activity by incorporation into the liposomes, and showed even in the detergent-solubilized state, its typical behaviors in both the unphotolyzed and photolyzed states. Upon solubilization, HsHR expressed in the E. coli membrane attains the proper folding and a trimeric assembly comparable to those in the native membranes.
AB - Salinarum halorhodopsin (HsHR), a light-driven chloride ion pump of haloarchaeon Halobacterium salinarum, was heterologously expressed in Escherichia coli. The expressed HsHR had no color in the E. coli membrane, but turned purple after solubilization in the presence of all-trans retinal. This colored HsHR was purified by Ni-chelate chromatography in a yield of 3-4 mg per liter culture. The purified HsHR showed a distinct chloride pumping activity by incorporation into the liposomes, and showed even in the detergent-solubilized state, its typical behaviors in both the unphotolyzed and photolyzed states. Upon solubilization, HsHR expressed in the E. coli membrane attains the proper folding and a trimeric assembly comparable to those in the native membranes.
KW - Archaeal rhodopsin
KW - Halorhodopsin
KW - Light-driven chloride pump
KW - Photocycle
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U2 - 10.1016/j.bbamem.2011.08.035
DO - 10.1016/j.bbamem.2011.08.035
M3 - Article
C2 - 21925140
AN - SCOPUS:80053366016
SN - 0005-2736
VL - 1808
SP - 2905
EP - 2912
JO - Biochimica et Biophysica Acta - Biomembranes
JF - Biochimica et Biophysica Acta - Biomembranes
IS - 12
ER -