期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 46, 期 1, 页码 47-55出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2005.07.006
关键词
heme biosynthesis; hemA; glutamyl-tRNA reductase; hemeprotein; cytochrome P450; 5-aminolevulinic acid
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, CYP17s, and mitochondrial CYP11A1. This new expression system provides a simple approach to obtain significant quantities of the active holo-form of recombinant hemeproteins. (c) 2005 Elsevier Inc. All rights reserved.
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