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Journal Article

Expression in Escherichia coli of c-type cytochrome genes from Rhodopseudomonas viridis

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Grisshammer,  Reinhard
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Oeckl,  Christine
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Michel,  Hartmut
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Grisshammer, R., Oeckl, C., & Michel, H. (1991). Expression in Escherichia coli of c-type cytochrome genes from Rhodopseudomonas viridis. Biochimica et Biophysica Acta-Gene Structure and Expression, 1088(2), 183-190. doi:10.1016/0167-4781(91)90053-O.


Cite as: http://hdl.handle.net/21.11116/0000-0006-FE54-E
Abstract
The genes coding for the photosynthetic reaction center cytochrome c subunit (pufC) and the soluble cytochrome c2 (cycA) from the purple non-sulfur bacterium Rhodopseudomonas viridis were expressed in Escherichia coli. Biosynthesis of the reaction center cytochrome without a signal peptide resulted in the formation of inclusion bodies in the cytoplasm amounting to 14% of the total cellular protein. A series of plasmids coding for the cytochrome subunit with varying N-terminal signal peptides was constructed in attempts to achieve translocation across the E. coli cytoplasmic membrane and heme attachment. However, the two major recombinant proteins with N-termini corresponding to the signal peptide and the cytochrome were synthesized in E. coli as non-specific aggregates without heme incorporation. An increased ratio of precursor as compared to ‘processed’ apo-cytochrome was obtained when expression was carried out in a proteinase-deficient strain. Cytochrome c2 from R. viridis was synthesized in E. coli as a precursor associated with the cytoplasmic membrane. An expression plasmid was designed encoding the N-terminal part of the 33 kDa precursor protein of the oxygen-evolving complex of Photosystem II from spinach followed by cytochrome c2. Two recombinant proteins without heme were found to aggregate as inclusion bodies with N-termini corresponding to the signal peptide and the mature 33 kDa protein.