Functional Expression and Characterization of a Bacterial Light-harvesting Membrane Protein in<i>Escherichia coli</i>and Cell-free Synthesis Systems

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  • SHIMADA Yuichiro
    Department of Biomolecular Engineering, Graduate School of Engineering, Center for Interdisciplinary Research, Tohoku University
  • WANG Zheng-Yu
    Department of Biomolecular Engineering, Graduate School of Engineering, Center for Interdisciplinary Research, Tohoku University
  • MOCHIZUKI Yushi
    Department of Biomolecular Engineering, Graduate School of Engineering, Center for Interdisciplinary Research, Tohoku University
  • KOBAYASHI Masayuki
    Department of Biomolecular Engineering, Graduate School of Engineering, Center for Interdisciplinary Research, Tohoku University
  • NOZAWA Tsunenori
    Department of Biomolecular Engineering, Graduate School of Engineering, Center for Interdisciplinary Research, Tohoku University

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  • Functional Expression and Characterization of a Bacterial Light-harvesting Membrane Protein in Escherichia coli and Cell-free Synthesis Systems

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Heterologous expression of a bacterial light-harvesting (LH) integral membrane protein was attempted using Escherichia coli cells and cell-free synthesis systems prepared from E. coli extracts. The α-apoprotein of LH1 complex from purple photosynthetic bacterium Rhodospirillum rubrum was overexpressed as a recombinant protein with a histidine (His6) tag added to the carboxyl terminus. Both of the expression systems produced α-apoprotein in a fully functional form as can judged by its ability to form a structural subunit with native β-apoprotein and the pigment molecule bacteriochlorophyll a. The expression product in E. coli appears to be located in the inner cell membrane and can be almost completely extracted by 0.5% (w/v) Triton X-100. Circular dichroism measurement indicated that the expressed α-apoproteins from both systems had α-helical contents essentially identical with that of the native one. About two thirds of the α-apoprotein expressed in E. coli was found to have the amino terminal methionine residue modified by a formyl group. About one third of the α-apoprotein expressed in the cell-free system was found to be oxidized at the side chain of the amino terminal methionine residue. Functional expression of the α-apoprotein using the cell-free system provides an useful example for producing highly hydrophobic integral membrane proteins with relatively large quantities sufficient for biophysical and structural analysis.

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