title: Functional expression of a two-transmembrane HtrII protein using cell-free synthesis.
abstract: An approach of cell-free synthesis is presented for the functional expression of transmembrane proteins without the need of refolding. The transmembrane region of thepharaonishalobacterial transducer protein,pHtrII, was translated with various large soluble tags added (thioredoxin, glutathione S-transferase, green fluorescentprotein and maltose binding protein). In this system, all fusionpHtrII were translated in a soluble fraction, presumably, forming giant micelle-like structures. The detergent n-dodecyl-β-d-maltoside was added for enhancing the solubilization of the hydrophobic region ofpHtrII. The activity of the expressedpHtrII, having various tags, was checked using a pull-down assay, using the fact thatpHtrII forms a signaling complex withpharaonisphoborhodopsin (ppR) in the membrane, as also in the presence of a detergent. All taggedpHtrII showed a binding activity withppR. Interestingly, the binding activity withppR was positively correlated with the molecular weight of the soluble tags. Thus, larger soluble tags lead to higher binding activities. We could show, that our approach is beneficial for the preparation of active membrane proteins, and is also potentially applicable for larger membrane proteins, such as 7-transmembrane proteins.
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan; PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Sudo
authors.affiliation: Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo 194-0031, Japan.
authors.forename: Rikou
authors.initials: R
authors.lastname: Tanaka
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
authors.forename: Toshitatsu
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: College of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Kamo
authors.affiliation: Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo 194-0031, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Kohno
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan; Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Chojiro
authors.initials: C
authors.lastname: Kojima
keywords: cell-free protein synthesis; membrane protein; protein-protein interaction; sensory rhodopsin