I'm having problems expressing human proteins in E.coli. I seem to fall out in expressing the protein; I use E.coli BL21(DE3) and am currently trying to express human P450 and P450 reductase as well as some other proteins. P450s genes I try to express from are usually in pCWori vector (pCW). I find that transforming and expressing protein from this vector is difficult. I do not have a map of the vector and please note, I get pink colonies when expressing the protein with 0.5 mM IPTG and with 100 microgram/ml ampicillin. I know that the vector has Amp resistance gene, but there is no idea of how I can know if the P450 enzymes have been expressed, except for carbon monoxide binding assay (CO-difference spectra). Since our heme and flavo- proteins are actually membrane proteins, the expressed proteins are expected to localize in the inner membrane. There seems to be a mysterious reddish-pink colour in the pellet when I collect cells after they are induced with IPTG (temp. is usually reduced to 28-30 degrees) and then culturing for 24-48 hrs. I wish to know if someone has observed change in colour of cells induced with IPTG. Is this an indication of expression? I added delta-aminolevulinic acid (d-ALA, a heme ring precursor) at 0.5 mM and still, no expression for CYP450 vector transformed cells; conversely, pBTR1 vector with cytochrome c expressed in DH5 alpha without the addition of d-ALA. Please help me with ways to achieve CYP450 heterologous expression.
Edited by dnlndrw, 13 July 2011 - 12:48 AM.