GST-protein purification: degradation product - (Apr/14/2007 )

Hi,

I'm trying to purify GST-tagged proteins, but have problems with GST-containing degradation products that show up just as strong as my target protein.
What I do:
use BugBuster Master Mix to lyse cells
keep samples on ice and centrifuge at 4C (I do not use a cold room and washing and elution buffer are not on ice)
add AEBSF (1mM) as well as proteinase inhibitor (sigma with AEBSF 1mM, too) to my lysis buffer

Any ideas, how to get rid of that degradation product?

Thanks,
Krisztina

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What's the size of the target protein and the contaminant?

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Also, how long did you wash and elute? It would be best to use cold washing and elution buffer.

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HI,

The target protein is about 37kDa, the contaminant (which is GST positive) is around 25kDa

Thanks for your help

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Hi,

The washing was 3X 5 min spinning at 4C. Elution was 1hr RT followed by overnight 4C and a final elution 1 hr RT (3 eluates).
I've tried keeping everything on ice all the time (except elution) and everything was prechilled, event the stripettes and pipette tips (so washing was with cold buffer centrifuging at 4C). The contamination was a bit less, but still strong and happy. Do you think cold elution could eliminate it completely?
Another strange thing I observed: using BugBuster to lyse cells, after 10 min the lysate was a clear liquid. However, during the overnight incubation with the sepharose beads at 4C, the lysate became cloudy (started to change after about 6 hrs). Can it be a problem with the lysis?
Anyway, thanks for your tips.

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Hi, I have a few suggestions:

1) Make sure that you have not also transformed vector with no insert. It is sometimes possible that you can pick what you think is one colony from a plate and actually get two. When you go to purify the plasmid you have a mixture of plasmids one with the insert and one without it. This would explain the 25kDa protein which is GST. To check for this, retransform into DH5alpha and repurify the plasmid.

2) Alternatively, the cleavage or truncated protein already has accumulated inside the bacteria before lysis and there is nothing you can do about it. You can check this by loading some cells onto SDS-PAGE without breaking them. IMO its unlikely the cleavage occurs after the lysis during purification since you have the correct inhibitors. If this is the case, ion exchange should be sufficient to separate the GST from your fusion protein. Since you have already purified your protein of interest, I would clean it this way. Choose a pH where you fusion will stick to the ion exchange column and the GST will flow through.

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I normally try to do as fast as possible, so the elution step I never do overnight. However, how did you induce your protein? Is it possible to run a complete set of sample to check which step degradation occurred, i.e. uninduced bacteria, induced bacteria, lysate (after homogenization), supernatant (after spin), pellet (after spin) and eluted samples? It may give you better idea.

Also, if degradation happened in the bacteria, you may have to use other protocols to reduce it, e.g. overnight express system, or 30oC induction...

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Elution overnight is fine...there are no proteases by the time you are eluting anyway if you have properly washed the column. I wouldn't do it either but it principle there shouldn't be a problem.

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Hi,

That is a good idea. I am checking at the moment if degradation can occur in the cell. Thanks.

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Thanks for the idea, I am currently doing this checking. I tried LB media+IPTG induction overnight at RT and also a so called MagicMedia from Invitrogen that needs no induction and it was for 24hrs growing at RT. Do you think it would be a good idea to reduce the growing period or even more the temperature after induction? can it reduce degradation if I do less expression (and compensate it with more volume)?
Anyway, thank you very much for your help.

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