Hi guys,
I am encountering a problem that my protein can't be purified by affinity purification, namely GST resin. This protein is a mutated protein where 20 amino acids were deleted. My protocol is: after bacteria BL21 DE3 growing at 37 (200mL culture in 500 mL flask)until OD600 reaches 0.8, and then add 1mM IPTG and switch to 30 degree overnight. The wild-type protein was expressed and purified pretty well, but the mutated protein can't be purified. Did my protein go into inclusion bodies? because I found that after sonication, the solution of wild-type and mutated proteins were still not clear, but GST empty vector was very clear. If my mutated protein went into inclusion bodies, so what's wrong with my wide-type protein?
can somebody help me? Thanks a lot!
Cheers
Two potential problems are: low expression of the mutant or good expression but gong to inclusion bodies. Is it any way to measure expression before purification (by Western?)?
If going to inclusions bodies you may check to lower IPTG and/or lower temperature to give opportunity for the protein to fold correctly.
Hi there,
I don't understand : the wt protein is properly expressed and purified as a GST fusion, yes or no?
So the wt fusion has been correctly purified, right? So what you should do is to compare step by step what you had with the wt and what you get with the mutant (comparative SDS/PAGE and GST WB confronting wt and mutant samples like total lysate, cleared lysate, pellet lysate) to try to know where the mutant protein is and first if it is actually expressed...
Hi Feng,
A quick question: what aprt of the GST sequence did you delete? Any such large deletion (20 amino acid residues equals 60 bp on the DNA) may have a profound effect on the GST stabilty and/or functionality I'm afraid.
Best regards,
Philippe
Ah OK. So your wt protein is expressing at detectable amounts in BL21DE3, but the fusion with GST (in C-term I presume) is not.
You should try, as previously suggested, to grow you E coli at room temperature and not at 30°C for the induction. It frequently helps when facing a no expression problem.
Best regards,
Philippe
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