The development of an efficient system for heterologous expression of cytochrome P450s in Escherichia coli using hemA gene co-expression.
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.
Harnastai
Ivan N
IN
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevicha str.5, Minsk 220141, Belarus.
Gilep
Andrei A
AA
Usanov
Sergey A
SA
D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005822:Genetic Vectors; D006418:Heme / Q000096:biosynthesis; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism; D013255:Steroid 21-Hydroxylase / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D013255:Steroid 21-Hydroxylase; D013254:Steroid 17-alpha-Hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase