GST-Fusion problems

Andy Scotter pcxajs at unix.ccc.nottingham.ac.uk
Fri Feb 25 09:45:06 EST 2000


	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?

	Cheers in advance

	Andy

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Andy Scotter
Department of Chemistry
University of Nottingham
University Park
Nottingham
NG7 2RD
0115 9514193
pcxajs at unix.ccc.nottingham.ac.uk

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