I have recombinant protein 30 kDa and Nus-tagged 68 kDa. After induction i found 98 kDa and 68 kDa in induced sample. why transformant E. coli BL21 Star produced incomplete protein? recombinant plasmid ~ 8000 bp
Dear Prawit
is it possible that the linker beetween tag and protein is exposed to protease action and you can have protein degradation. Its happened to me several time in the past using n term gst fusion .
i think that first of all you can try ti repeat the expression using lower induction temperature to see if in this way protease are less active and degradation is reduced. otherwise you can change e.coli strain, the fusion tag or the linker beetween the tag and the protein but in some cases, exprcially if you usato observe partial degradation, the optimization of culture condictions (and of course use of protease inhibitors in the lysis buffer) can solve your problem
good luck
ciao
Manuele
Dear, Manuele
Thank you, i will try at low temp or change bacteria.
Did you find your recombinant protein or find only tag after protein degradation?
I tag 6xHis at N and C. I found intensive ban of 68 kDa and fade ban of 98 kDa in eluted fraction after purification, but i not found 30 kDa of protein degradation.
Dear Prawit, I agree that it's a possibility that the two bands are from degradation of your fusion protein, but it seems to me that the 68kD and 98kD species may not be from degradation of your protein, since you see the same two species when you use the 6xHis tag. So, those two bands could be unrelated bacterial proteins that bind non-specifically to your affinity resin. You can do a western blotting assay of whole cell extracts to be sure your protein is expressed. If it's expressed at all, you can test other expression conditions as Manuele suggested. Good luck with your experiments.
Hi Prawit,
I agree with Hua Xiao, please first use WB to prove that your target protein is indeed expressed. If there is a TEV restriction site between the target protein and the tag, you can perform preliminary verification by enzyme digestion test. If it turns out that your recombinant protein has been degraded, it is recommended to add enough protease inhibitors during the purification process or to try different tags, truncations, and expression systems.
All the best!
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