GST-Fusion problems

Anton Tutter atutter at aim.salk.edu
Sun Feb 27 20:21:25 EST 2000


in article Pine.SOL.4.05.10002251437200.15426-100000 at granby, Andy Scotter at
pcxajs at unix.ccc.nottingham.ac.uk wrote on 2/25/00 6:45a:

> I'm trying to purify a GST fusion protein using glutathione
> sepharose 4B following the batch purification method.  The protein fused
> to GST is only 1.6KDa compared to GST which is about 27KDa.  My control
> induction samples where I should be getting just GST produced give a
> larger band at about 40-45KDa instead of 27KDa and my fusion protein is in
> the same place too. I don't get any dense bands at the ~27kDa position.
> Upon purification I get bands at 27-29KDa as expected for the pure fusion
> and GST but also the same >40KDA bands.
> I don't think the fusion proteins is dimerising as it is a denaturing gel
> so what could the dense band be.
> It should be GST produced from the IPTG induction of pET42 in BL21(DE3)
> pLysS in the case of the control and my fusion protein in my other
> inductions using pET42 with an insert between the BamH1 and EcoR1 sites.
> Any ideas or advice for GST-fusion purification?
> 

i know there is a 40kDa band that always co-purifies with my GST-fusion
preparations.  according to Pharmacia, it is a bacterial heat shock protein
that commonly co-purifes. i have even run my preparations over further
columns such as Q-sepharose or S-sepharose and it still co-purifies.

--anton






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