I have cloned MAPK3 in BL21 cells. I have already tried protein induction at 37, 28 and 22 degree Celsius with IPTG conc ranging from 0.4mM to 2 M. But I got no protein induction. I have also checked the frame twice. so there is no issue in that. please give your suggestions so that I can get the desirable results. right now I am not able to know which are the loopholes. thank you.
Hello! Did you check for codon usage? This is the standard website for this, https://www.kazusa.or.jp/codon/ There is also the graphical codon usage analyzer, http://gcua.schoedl.de/ which makes nice pictures, and can import the data from kazusa. If you don't see anything wrong, then it may be that your protein requires some extra help folding, or perhaps a post-translational modifcation which indicates you may need baculoviral or pichia pastoris as a host organism for your protein expression, or even CHO cells. There are a horde of N-linked or O-linked glycosylation predictors that will indicate that E. coli isn't the best system. many at ExPASy or CBS bioinformatics. Hopefully, you are testing both the cell supernatant and cell pellet by western blotting? Your protein could be expressed into inclusion bodies. I hope any of this helps!! Good luck!
Can you provide the sequence of the plasmid you constructed to express it? If you can't, what's the plasmid backbone?
Hi Saumya,
in addition to optimising codons one can also try other (DE3) strain. Is it absolutely excluded that your BL21 cell are not (DE3)?
Alternatively, you can change the expression vector (there are plenty of them: pET series, pGEX series, pLATE series, etc)
Best of luck! :)
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