No Protein Induction - (Mar/02/2005 )

Hi everybody. I have been trying to overexpress and purify an active enzyme from S. aureus in BL21(DE3). These previous attempts were of a soluble, but truncated version of my protein. However, I have had no luck trying to overexpress a new construct of this enzyme which contains the entire length of the protein, transmembrane (hydrophobic) regions and all. I would assume that the transmembrane region would only hinder purification, not overexpression. I have looked at the idea of rare codon usage, and that does not seem to be an issue with this protein. Any ideas? Thank you!

It appears that your problem belies in the inclusion of the hydrophobic stretch in your protein. As you proein is a memebrane protein, it doesn't mean that when you overepxress it, it automatically expresses as a emebrane protein. It requires appropriate signal peptides.

If you are trying to express as a soluble then, the protein might have aggregated into inclusion bodies. first chack if atall the protein is expressed. Run the cell extract/homogenate of induced and uninduced clones on SDS PAGE and check for expression. If you find the band then, but no activity in the relevant sample, then the protein is aggregating into inclusion bodies.

You can try to dissolve the inlcusion bodies with urea or guanidium.HCL and reanture it. During reanturation, inlcude different types ofdetergents and check for stability. Some kits for solubilization and stabilization are available from HAMTON RESEARCH. These are meant for use during crystallization; however, the readymade buffers and solutions give preliminary conditions to start with.

Thank you for you help. The issue is not really activity at this point; I don't even see induction on the gel so I haven't been able to attempt purification. I am going to try taking aliquots at shorter inductions times to see if maybe the protein is being degraded?

hi
as you said your protein is a membrane protein, do you extract your protein well (if supposing there's a expression)?

Fred