GST expression problem - low fusion, high GST alone (Apr/02/2008 )

I am trying to purify a GST-fusion protein for a kinase assay. I have successfully cloned my gene into pGEX 4T2 and sequencing confirms that is in in-frame with the GST and the stop codon. When I purified my protein on GSH-agarose I found that while I was able to purify a small amount of my GST-fusion (correct size, recognized by a insert-specific antibody) but by coomassie the vast majority of product was GST alone. When I tested my induction I see that I am indeed getting a ton of GST production and very little GST-fusion induction.

Does anyone know what could be causing this? I've heard of codon bias, and this is a murine protein so that may be a possibility. My DNA is purified and clean, so there should not be any empty pGEX being transformed into the BL-21s. I normally don't screen my colonies since I am transforming with purified DNA, is this a common problem and I just need to screen a bunch of clones for induction of the fusion?

Thanks for any help.

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It's possible that you could be getting cleavage of your protein, especially if this GST clone has a thrombin cleavage site in it. I found this info on the web. Hope it helps.

Some think that the engineered thrombin
cleavage site or some other cleavage site within the recombinant protein might
be recognized by an unidentified protease present in E. coli lysates, and that
this is especially evident in the E. coli strain BL21. To avoid this kind of
protease activity, netters suggest that the following precautions can help
reduce the cleavage:

(1) Use a relatively fresh transformant made using plasmid DNA stored in the
freezer. The bacteria should be no more than one month old and should not be
streaked from glycerol stocks because this has been found to be unreliable for
proper synthesis of GST fusions. Also, several different strains of E. coli
should be used.

(2) Grow the E. coli in the presence of 0.25-0.5% glucose to keep expression
from the tac promoter to a minimum before induction with IPTG.

(3) Grow the cells at the lower temperature of 22-30 degrees C. This will most
likely lower the yield of recombinant protein, but should lessen the activity of
the unknown protease and give a higher proportion of undegraded protein.

(4) Keep the induction time relatively short (2-4 hours).

(5) Include a protease inhibitor when lysing the cells.

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I used pgex-kt to make a fusion, screening numerous clones (for the clone and the empty vector). It was difficult to get alot of protein from my fusion, but I never had GST alone in that sample. Have you tried different induction times? What does your GST alone look like in comparison (or haven't you done that?) Also, what bug are you using? I used JM109. All worked well.

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I just did a western on my induction samples, and it looks like I am inducing a good amount of my fusion. I haven't run a GST-alone sample, but there is a distinct ~26kD band that shows up in my crude lysate after induction in a coomassie stained gel, and it is not recognized by an antibody for the inserted gene. I didn't alter induction time (~2 hours) but did alter IPTG concentration, and the induction of neither my fusion or the GST alone changes between the different concentrations (neither are present in un-induced bugs).

I'm thinking that this smaller GST band is present in a much higher molarity to my fusion, and is binding up most of the GSH-agarose, preventing me from purifying much of my fusion.

Is it possible to dialyze my lysate to remove that smaller protein and then purify on GSH-agarose? Maybe a quick "pre-clear" with GSH-agarose? Any other ways of filtering it out short of just IPing my fusion out of the lysate?

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