In vivo und in vitro Expression von Membranproteinen am Beispiel archae- und eubakterieller Rhodopsine
Creators: name: Kalmbach, Rolf
Publisher: name: Technische Universität Dortmund
Year: 2005
Subjects: subject: In vitro Expression
subject: Zellfreie Synthese
subject: Liposomen
subject: Archaebakterien
subject: Eubakterien
subject: Rhodopsine
subject: G-Protein gekoppelte Rezeptoren
subject: GPCRs
subject: Bakteriorhodopsin
subject: Proteorhodopsin
subject: Black Lipid Membrane
subject: BLM
subject: Laserblitzphotolyse
subject: Photozyklus
subject: In vitro expression
subject: cell-free synthesis
subject: liposomes
subject: archaebacteria
subject: eubacteria
subject: rhodopsins
subject: G-protein coupled receptors
subject: bacteriorhodopsin
subject: proteorhodopsin
subject: black lipid membrane
subject: laserflash-photolysis
subject: photocycle
subject: 610

description: The completion of the human genome project and the development of sensitive high-throughput assay techniques initiated a dramatic acceleration in the pace of biological research. An essential prerequisition for further progress will be the establishment of effective and selective methods for the expression of the gene products in their functional state. In this work the cell-free protein biosynthesis of integral membrane proteins was established. Several rhodopsins from archae- and eubacteria (bacteriorhodopsin bR, N.pharaonis halorhodopsin NphR, N.pharaonis sensory rhodopsin II NpsRII and proteorhodopsin PR) as well as the G-protein coupled human Glycinreceptor (hGlyR1) were successfully synthesised in a coupled in vitro transcription/translation system based on E.coli cell extracts. Insertion of the proteins in an artificial hydrophobic environment consisting of various detergents or lipids was screened by proteolytic digestion of protease-accessible regions. bR and to less amounts NpsRII and PR were inserted into small unilamellar liposomes consisting of phospholipids in the fluid L-phase. The estimated protein yield of cell-free synthesised bR (bRcf) was 20 mg/liter. Functionality of the cell-free synthesised bRcf was confirmed by laserflash-photolysis. The photocycle of bRcf is similar to that of the native protein. The M-Intermediate decays with a slower rate constant than bR in the purple membrane but comparable with polarized cell-envelope vesicles from H.salinarum. The photocurrent of bRcf could be meassured directly using the crude reaction mixture. The high current of 1 nA/cm2 can be explained by the unidirectional orientation of the protein in the liposomes. To compare functional in vitro with in vivo expression of membrane proteins the heterologous in vivo expression was applied to the bR-like PR from uncultivated marine eubacteria. PR was functionally expressed and purified from E.coli cells in yields up to 9 mg/liter culture volume. PR belongs to the family of retinal-containing seven-transmembrane helix receptors. After irradiation PR cycles through subsequent steps of spectrally distinct intermediates. This photocycle results in the transport of a proton across the cell membrane. The absorption maximum of PR is dependent on the pH and shifts from 518 nm at alkaline to 539 nm at acidic pH. Based on a detailed kinetic analysis of the transient absorption changes of the alkaline and acidic species, two distinct photocycle models were derived. The photocurrents of PR were investigated by the black lipid membrane technique, revealing that PR pumps protons outwardly at alkaline pH, but the pumping direction is inverted at acidic pH. These results show that PR acts as an efficient proton pump suggesting a primary role in bacterial energy conversion. The existence of two competing photocycles with inverse pumping directions could allow for a more subtle regulation of the membrane potential. Regarding the fact that marine bacteria contribute considerably for the biomass on earth, PR phototrophy might be a globally significant oceanic microbial process beside the photosynthesis reaction.