PMID:16122943
| Citation |
Harnastai, IN, Gilep, AA and Usanov, SA (2006) The development of an efficient system for heterologous expression of cytochrome P450s in Escherichia coli using hemA gene co-expression. Protein Expr. Purif. 46:47-55 |
|---|---|
| Abstract |
Biosynthesis of heme in Escherichia coli is under strict regulatory control since free heme or intermediates of its biosynthesis are potentially toxic for the cell. Under normal physiological conditions a bacterial cell does not have significant levels of free heme. Recombinant hemeproteins with affinity for heme lower than that of intrinsic cell proteins are often only isolated as apo-proteins. Moreover, for a number of hemeproteins expressed as apo-protein in E.coli it is not possible to reconstitute holo-protein in vitro. To circumvent these issues, fully active recombinant hemeproteins are usually expressed with expensive 5-aminolevulinic acid supplementation. In the present work, we construct the helper plasmid pHg expressing glutamyl-tRNA reductase (hemA) a key enzyme catalyzing the rate-limiting reaction in heme biosynthesis in E. coli, to avoid the necessity of 5-aminolevulinic acid supplementation. Overexpression of HemA restores the proper balance between protein and heme synthesis so that the newly synthesized recombinant apo-protein is continuously converted to holo-protein. The pHg plasmid is capable of supporting high-level expression of microsomal CYP1A1, CYP1A2, CYP21, CYP17, and mitochondrial CYP11A1. This new expression system provides a simple approach to obtain significant quantities of the active holo-form of recombinant hemeproteins. |
| Links |
PubMed Online version:10.1016/j.pep.2005.07.006 |
| Keywords |
Aldehyde Oxidoreductases/genetics; Aldehyde Oxidoreductases/metabolism; Cholesterol Side-Chain Cleavage Enzyme/genetics; Cholesterol Side-Chain Cleavage Enzyme/metabolism; Cloning, Molecular; Cytochrome P-450 CYP1A1/genetics; Cytochrome P-450 CYP1A1/metabolism; Cytochrome P-450 CYP1A2/genetics; Cytochrome P-450 CYP1A2/metabolism; Cytochrome P-450 Enzyme System/genetics; Cytochrome P-450 Enzyme System/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/growth & development; Escherichia coli/metabolism; Genetic Vectors; Heme/biosynthesis; Kinetics; Mitochondria/enzymology; Mutagenesis; Recombinant Fusion Proteins/metabolism; Restriction Mapping; Steroid 17-alpha-Hydroxylase/genetics; Steroid 17-alpha-Hydroxylase/metabolism; Steroid 21-Hydroxylase/genetics; Steroid 21-Hydroxylase/metabolism |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.