Protocol

Heterologous Gene Expression in E.coli

Volume 705 of the series Methods in Molecular Biology pp 123-150

Date:

Expression of Recombinant Cytochromes c in E. coli

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Abstract

Answering questions about proteins’ structures and functions in the new era of systems biology and genomics requires the development of new methods for heterologous production of numerous proteins from newly sequenced genomes. Cytochromes c – electron transfer proteins carrying one or more hemes covalently bound to the polypeptide chain – are one of the most recalcitrant classes of proteins with respect to heterologous expression because post-translational incorporation of hemes is required for proper folding and stability. However, significant advances in expression of recombinant cytochromes c have been made during the last decade. It has been shown that a single gene cluster, ccmA–H, is responsible for cytochrome c maturation in Escherichia coli under anaerobic conditions and that constitutive co-expression of this cluster under aerobic conditions is sufficient to provide heme incorporation in many different types of cytochromes c, regardless of their origin, as long as the nascent polypeptide is translocated to the periplasm. Using conditions that result in sub-maximal protein induction can dramatically increase the yield of mature protein. The intrinsic peroxidase activity of hemes can be used as a highly selective and sensitive detection method of mature cytochromes in samples resolved by gel electrophoresis.

Key words

Cytochrome c Escherichia coli heterologous expression periplasmic expression peroxidase reaction UV–Vis spectroscopy