I have expressed my protein of interest in E.coli and purified it via the Ni-NTA column. Then did a UV-VIS spectroscopy to see if it is binding to its chromophore. Even after several trials, it was not binding. But in the in-silico simulations, we could see that the chromophore binding pocket is present and it is stable. I would like to know what could be the potential reasons and how can I overcome it.
Dear Alisha Bhanu Pattani Ameerjan
is not so easy to answer to your questions before know more details about the protein and the cofactor.
Just 2 questions:
1) do you know if your protein is predicted to contains structural S-S bonds that can be not correclty assembled in E.coli?
2) Do you know if the cofactor have to be assembled during the protein folding?
Because in this case could necessary supply the cofactor in the e.coli culture media and maybe move to periplasmic expression to improve the probability the cofactor could be inserted to the protein.
Each protein is different the the result it may depends from the cofactor size, from the the location of the cofactor binding site (is it surface exposed or buried in the protein core?)
best regards
Manuele
Dear Manuele,
Thank you for the quick response. So protein is cryptochrome protein and its cofactor is FAD. The other isoform of this protein was shown to bind to FAD. This isoform (which is my protein of interest) only has
one extra exon of 29 amino acids which is located far away from the FAD binding pocket. The answer to both of your questions are currently unknown as this is the first time expressing this protein.
It could be that your purification conditions are stripping the protein of its cofactor, or disrupting its structure, removing its ability to bind the cofactor.
You could try to include the co-factor during the expression, during the purification, or even denaturing and refolding in the presence of the cofactor.
Dear Manuele Martinelli
After some discussions regarding your suggestion, I am planning to do a periplasmic expression of my protein. Do you have a protocol for the same and which vector have you tried and what worked best for you? Do you think pET-22b(+) with PeIb signal sequence is a good option?
DEar Alisha Bhanu Pattani Ameerjan
I never used the pet22b, but in the past i used pet20b that is quite similar and it worked well for some proteins.
In other cases, for example for production of Fab antibodies in the bacterial periplasm, i replaced by PIPE cloning the pelB leader with the ompA leader but the differences were limited.
I suggest to you to:
- test different induction temperatures, also lower (eg 22-25C) sInce periplamic expression require protein traslocation, is important to not pump to much the expression bacause you can saturated the traslocation system and the T7 promoter is very strong.
- supplement the culture media with FAD
good luck
Manuele
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