High Yield of Methylophilus methylotrophus Cytochrome c″ by Coexpression with Cytochrome c Maturation Gene Cluster from Escherichia coli |
| |
Authors: | Nicholas J. Price Lorraine Brennan Tiago Q. Faria Erik Vijgenboom Gerard W. Canters David L. Turner Helena Santos |
| |
Affiliation: | a Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-156, Oeiras, Portugal;b Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands;c Chemistry Department, University of Southampton, Highfield, Southampton, SO17 1BJ, United Kingdom |
| |
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 1H-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. |
| |
Keywords: | |
本文献已被 ScienceDirect 等数据库收录! |
|