I have a codon optimized construct for a viral gene and I am expressing it in BL21DE3 strain. for all other papers mentioning its purification it is coming in soluble fraction but for me it is coming in inclusion body. I have tried various IPTG concentrations and even various temperatures also, like 37 degree, 18 degree. What should I do to get it in soluble fraction?
Stay close to published conditions, contact the authors if you suspect that the method is incomplete.
Test the expression in other BL21-DE3 strains : Gold, Rosetta, C41, etc. In case your protein precipitates during lysis you can increase salt concentration (>500mM NaCl), change pH, add detergents (0,05% Triton X-100) and glycerol (up to 10%).
Is the mentioned viral protein ,codon optimized in the publications you are referring to?
How did the other groups detect the protein? Did they see an overexpressed band on SDS-PAGE or was it detected by western blotting. Tiny quantities can be detected by western and not very useful for large scale protein studies. Is there any activity that you can check? Did the previous publications present robust data using this purified protein?
Good Luck
I am in agreement with what everyone has said but if you are certain you are following the published protocol to the letter then it must be something subtle that they were doing that they did not report. One thing to consider is the point of harvesting the cells. We have found in one or two cases that even, at 18degC, if the harvesting of cells is delayed by only one or two hours the protein goes from soluble to inclusion bodies. Try different harvesting times. Also one other thing to try is to use autoinduction which does not use IPTG but a glucose/glycerol/lactose mixture so induction is is acieived slowly, this may improve inclusion body problem
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