Functional expression and characterization of a bacterial light-harvesting membrane protein in Escherichia coli and cell-free synthesis systems.
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 alpha-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 alpha-apoprotein in a fully functional form as can judged by its ability to form a structural subunit with native beta-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 alpha-apoproteins from both systems had alpha-helical contents essentially identical with that of the native one. About two thirds of the alpha-apoprotein expressed in E. coli was found to have the amino terminal methionine residue modified by a formyl group. About one third of the alpha-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 alpha-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.
Shimada
Yuichiro
Y
Department of Biomolecular Engineering, Graduate School of Engineering, Center for Interdisciplinary Research, Tohoku University, Sendai, Japan.
Wang
Zheng-Yu
ZY
Mochizuki
Yushi
Y
Kobayashi
Masayuki
M
Nozawa
Tsunenori
T
D001426:Bacterial Proteins / Q000235:genetics; D002474:Cell-Free System / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D012247:Rhodospirillum rubrum / Q000737:chemistry; D013057:Spectrum Analysis
D001426:Bacterial Proteins; D045342:Light-Harvesting Protein Complexes