High yield of Methylophilus methylotrophus cytochrome c by coexpression with cytochrome c maturation gene cluster from Escherichia coli |
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Authors: | Price N J Brennan L Faria T Q Vijgenboom E Canters G W Turner D L Santos H |
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Institution: | Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal. |
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Abstract: | Heterologous expression of c-type cytochromes in the periplasm of Escherichia coli often results in low soluble product yield, apoprotein formation, or protein degradation. We have expressed cytochrome c from Methylophilus methylotrophus in E. coli by coexpression of the gene encoding the cytochrome (cycA) with the host-specific cytochrome c maturation elements, within the ccmA-H gene cluster. Aerobic cultures produced up to 10 mg holoprotein per liter after induction with IPTG. In the absence of the maturation factors E. coli failed to produce a stable haem protein. Cytochrome c" isolated from the natural host was compared with the recombinant protein. No structural differences were detected using SDS-PAGE, UV-Visible spectroscopy, differential scanning calorimetry, and (1)H-NMR spectroscopy. The success in expressing the mature cytochrome c in E. coli allows the engineering of the cycA gene by site-directed mutagenesis thereby providing an ideal method for producing mutant protein for studying the structure/function relationship. |
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