Dear all
My target gene was inserted into pGAPZα and was successfully verified by sequencing. Then constructs were digested with AvrII , BspHI, and Bglll seperately. Each of these lineaized constructs was transformed into Pichia (widle type). However, when I got up to a point of expressing the protein in a small batch and verification using SDS-PAGE and Western blot, I couldnt get any expression. I have been trying for several months in order to express it even with the inclusion of GFP after the insert gene.
Since this problem occurs within the batch set up, perhaps the batch conditions that you maintain might not be sufficiently driving the protein formation within the cells. It could also be a case by which the immediate batch environment denatures the protein irreversibly no sooner its formed.
Biotic stress within system?
Have you lysed the cells and carried out western blot on the cytosol? Use a sample of empty vector-containing P. pastoris as a control, that will tell you if your protein is being produced but not passing through the cell membrane.
If that's the case, you could being altering the signal sequence. We sometimes include both the native and the recombinant signal sequences for expression in Pichia. Other things you could try include reducing the temperature to 25 C upon induction.
Pichia is a great expression system, but occasionally not suitable for expression of certain genes. You may have to look as a more suitable expression host.
Hope this helps!
Western blotting can sometimes require optimization to see a protein band at times..Things to try:
1) Try silver staining which can detect low yield protein - try this with untransformed and transformed construct side by side
2) Redesign your construct by truncating disordered regions in the protein
3) Maltose binding protein (MBP) has proven useful for expression and solubility
4) I have never used Pichia Pastors, but if you can try selecting for stable cell lines
Hi,
are you sure you have an expression problem? Did you check if your gene is actually integrated into the Pichia genome? Invitrogen provides PCR primer sequences for this purpose. Or you could try using higher zeocin concentrations to rule out an error during the selection.
I am also trying to express a protein intracellularly. I confirmed gene insert by PCR and did RT PCR to check mRNA. Got both positive, but no bands on SDS PAGE and Western blot!
Thank you guys very much for your tips. I followed all your tips throughly and I got it shown on Western blot. One more Thing that I added ( and might be useful for Aniu Parokkaran) is loading highly concentrated proteins on SDS-page Gel and optimized Western blot.
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