At 5:56 PM +0100 8/26/10, Hari Namboodiri wrote:
Can anyone provide insights about expressing heme containing proteins in E.col? Does E.coli need any porphyrin precursor during expression or you need special E.coli strains. I have references mentioning delta-aminolevulinic acid for one ortholog but none for another. The enzyme is CYP51.
With E. coli, you definitely want to include 5-aminolevulinic acid (ALA) in the culture before induction. For membrane anchored CYPs you will probably also need to use a special vector to get good expression. Many have tried and failed to get expression of some CYPs using pET vectors. I suggest that you look into a vector like pCWori (see citation below).
- John Imai T, Globerman H, Gertner JM, Kagawa N, Waterman MR. Expression and purification of functional human 17 alpha-hydroxylase/17,20-lyase (P450c17) in Escherichia coli. Use of this system for study of a novel form of combined 17 alpha-hydroxylase/17,20-lyase deficiency. J Biol Chem. 1993 Sep 15;268(26):19681-9. PubMed PMID: 8396144. --
