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Problems purifying a protein expressed in Pichia


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#1 Pichia

Pichia

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Posted 15 September 2009 - 04:05 AM

Hi,
I was hoping that someone might be able to help me with a problem that I am having purifying a protein expressed in Pichia pastoris?

I cloned my gene into the pPICZalphaC vector for secretion in KM71 yeast, using the alpha factor secretion signal rather than the native signal peptide. Transformation was performed using the EasyComp Kit from Invitrogen; recombination of my gene of interest into the AOX1 site has been verified by PCR. A time-course experiment was performed to determine the optimum time for highest expression level of the protein following methanol induction. Western blots using both the anti-His and an antibody against my protein show expression and secretion into the media is fine.

However, when I come to purify the secreted protein from the Pichia supernatant (dialysed into Ni-NTA Binding Buffer, 300 mM NaCl, 50 mM NaH2PO4, 10 mM Imidazole), I see very inefficient binding to the Ni-NTA agarose i.e. most of my protein appears in the flowthrough from the column.

I have tried:
-Concentrating the supernatant prior to mixing with the agarose
-Increasing the time the supernatant is incubated with the agarose for, up to overnight, at 4 degrees C.
-reapplying flowthrough to the agarose multiple times
-reducing Imidazole in the binding buffer to 5 mM
-using a different batch of Ni-NTA agarose

All with no improvement in binding, which makes me wonder if the His-tag is somehow not available to bind for most of the protein that I am making (some protein does bind, and is purified very well, but relatively tiny amounts!).

For enzyme activity reasons, I donít want to purify under denaturing conditions.

I would be really grateful if anyone else has encountered similar problems and knows how to resolve it! Or if anyone has any suggestions?

Apologies for the long post and thanks for reading!




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