title: Mistic-fused expression of algal rhodopsins in Escherichia coli and its photochemical properties.
abstract: BACKGROUND
  Since algal rhodopsins, the eukaryotic seven-transmembrane proteins, are generally difficult to express in Escherichia coli, eukaryotic cells have been used for heterologous expression. Mistic, a membrane-associated protein that was originally discovered in Bacillus subtilis, has been shown to improve the expression levels of many foreign integral membrane proteins in E. coli when used as a fusion partner linked to the N-terminus of cargo proteins.
  
  
  METHODS
  Here, we expressed two algal rhodopsins with N- and C-terminal Mistic domains in E. coli-Acetabularia rhodopsin I (ARI) and Chlamydomonas sensory rhodopsin B (CSRB, channel rhodopsin 2). UV/VIS spectroscopy, pH titration of proton acceptor residue, laser-induced photolysis and electrophysiological measurement were used for investigating important residues in proton transport and spectroscopic characters of the proteins.
  
  
  RESULTS
  Protein yield of two algal rhodopsins was enhanced, obtaining 0.12mg of Mistic-ARI and 0.04mg of Mistic-CSRB per liter of culture. Spheroplast expression Mistic-ARI had outward proton-pumping activity, indicating protein functionality. Asp89 of ARI changed its protonation state by light absorption, and Asp100 was important for O(600) formation. Electrophysiology revealed that both residues took part in proton transport. The spectroscopic analyses of Mistic-CSRB revealed its characteristics.
  
  
  CONCLUSIONS
  Fusion to the membrane-integrating protein Mistic can enhance overexpression of eukaryotic type I rhodopsins in E. coli.
  
  
  CONCLUSIONS
  These findings indicate that Mistic fusion and E. coli expression method could be an effective, low cost technique for studying eukaryotic membrane proteins. This may have useful implications, for example, in studying structural characteristics and optogenetics for rhodopsins.
authors: Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Shinsu-Dong 1, Mapo-Gu, Seoul 121-742, Republic of Korea.
authors: Keon Ah
authors: KA
authors: Lee
authors: Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Shinsu-Dong 1, Mapo-Gu, Seoul 121-742, Republic of Korea.
authors: Sang-Soo
authors: SS
authors: Lee
authors: Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Shinsu-Dong 1, Mapo-Gu, Seoul 121-742, Republic of Korea.
authors: So Young
authors: SY
authors: Kim
authors: Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Shinsu-Dong 1, Mapo-Gu, Seoul 121-742, Republic of Korea.
authors: Ah Reum
authors: AR
authors: Choi
authors: Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Shinsu-Dong 1, Mapo-Gu, Seoul 121-742, Republic of Korea.
authors: Jung-Ha
authors: JH
authors: Lee
authors: Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Shinsu-Dong 1, Mapo-Gu, Seoul 121-742, Republic of Korea. Electronic address: kjung@sogang.ac.kr.
authors: Kwang-Hwan
authors: KH
authors: Jung
mesh_terms: D000076:Acetabularia / Q000737:chemistry*; D002696:Chlamydomonas / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry*; D010777:Photochemistry; D010940:Plant Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012243:Rhodopsin / Q000737:chemistry*
chemical_list: D008565:Membrane Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D012243:Rhodopsin
keywords: Algal rhodopsin; Bacteriorhodopsin; Ion channel; Proton transfer; Rhodopsin