Mistic-fused expression of algal rhodopsins in Escherichia coli and its photochemical properties

• Published in: Biochimica et biophysica acta Vol. 1850; no. 9; pp. 1694 - 1703
• Main Authors: Lee, Keon Ah, Lee, Sang-Soo, Kim, So Young, Choi, Ah Reum, Lee, Jung-Ha, Jung, Kwang-Hwan
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2015
• Subjects: absorption; Acetabularia - chemistry; algae; Algal rhodopsin; Bacillus subtilis; Bacteriorhodopsin; Chlamydomonas; Chlamydomonas - chemistry; electrophysiology; Escherichia coli; Escherichia coli - genetics; eukaryotic cells; heterologous gene expression; Hydrogen-Ion Concentration; Ion channel; membrane proteins; Membrane Proteins - chemistry; optogenetics; Photochemistry; photolysis; Plant Proteins - chemistry; Proton transfer; Recombinant Fusion Proteins - chemistry; Rhodopsin; Rhodopsin - chemistry; spectroscopy; titration; Bacteriorhodopsin; Rhodopsin; ChR2; Algal rhodopsin; Proton transfer; ARI; Ion channel; CSRB
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2015.04.002

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. 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. 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 O600 formation. Electrophysiology revealed that both residues took part in proton transport. The spectroscopic analyses of Mistic-CSRB revealed its characteristics. Fusion to the membrane-integrating protein Mistic can enhance overexpression of eukaryotic type I rhodopsins in E. coli. 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. •Fusion to the Mistic can enhance overexpression of eukarytic type I rhodopsins.•We used two Mistic domains to express two algal rhodopsins in E. coli.•The protein yields from E. coli were sub mg per liter of culture.•We first reported photochemical properties of purified Acetabularia rhodopsin I.