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[dnlndrw]

Hi,

   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.

[qzlabs]

dnlndrw, on 13 July 2011 - 12:45 AM, said:

Hi,

   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.

You did everything right. The color change upon the addition of IPTG indicated the expression of your cloned gene. If the construct prepared as you expected which can be confirmed by a DNA sequencing, the expressed protein must be whichever protein you cloned into the vector. If the gene in the vector is P450, the protein must be P450. You do not need worry too much about it. To confirm the expression, you can take two samples from uninduced (before IPTG) and induced cultures (after the induction with IPTG), and run them on SDS-PAGE to see if there is a band at P450 size only appeared in the induced sample. From what you described, your protein might be in the inclusion bodies. I would harvest cells after 12 hours maximum and change the temperature to room temperature after the addition of IPTG. Where you expect P450 to be? There is little inner membranes in a e.coli cell?
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