We have constructed GST-tagged protein to the N-terminal of GST in pGEX-4T2 plasmid which is about 70-90 KD. We tried different concentration of IPTG for induction but the expression is not enhanced. Also we tried at different temperature. Does anyone have a good suggestion?
Hi Zahir,
which E.coli are you using for expression?
In my experience BL21 (DE3) should work, but XL1blue did a better job for most GST-Fusions I expressed so far. Usually GST-Fusion proteins can be expressed in really nice quantities, so there might be something wrong with your construct.
Have these proteins been expressed in E.coli before from other constructs?
Have you sequenced the construct and checked that your reading frame is ok, no stop codon in cDNA e.g.?
Did you really fuse your protein-of-interest to the N-terminus of GST? If so you might need a flexible spacer to avoid misfolding of GST!
Best,
Kai
we have expressed this protein with His tag in BL21 and it was quite good but in GST-tag it is not expressed in BL21 and Rosetta strains. we tried with 0.05,0.1,0.5, 0.8 ans 1 mM IPTG at 18,25 and 37C, but still the expression is not observed at all.
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