I am expressing several genes using the pET28(a) vector in E. coli BL21 cells. While some recombinant proteins are expressed, they accumulate predominantly in inclusion bodies. Although I purify these proteins under denaturing conditions to assess their in vitro activity, the activity is lost during the purification process. What strategies could be employed to enhance the soluble expression of these recombinant proteins? Should I consider changing the expression vector or modifying the cloning strategy, such as selecting different restriction sites, to improve solubility and preserve functional activity?
Some common strategies to obtain a soluble protein are:
Alernatively, refolding the protein from purified inclusion bodies can be attempted.
Dear Akhileshwar Singh
The solutions suggested by Adam B Shapiro , expecially low temperature and fusion tag may allow to you to improve protein solubility in many cases, however it depends a lot from the properties of the protein that you are expressing:
Generally the protein are going in the IB for several reasons, e.g:
1) Are membrane proteins;
2) Are protein rich of cysteins forming structural disulfides that cannot be formed in the bacterial citoplasm since the enviroment is too reducing;
3) Are enzimes with toxic activity for the E.coli;
4) Are enzimes that contain cofactors (e.g metals) essential for their structural stability that are not aviable in enough amount;
Reduction of induction temperature is a possible general rolu
Each of this problem may be solved in a different way eg:
- periplasmic expression or origami strain for disulfide formation in E.coli or move to mammalian expression sistem, eg Expi293;
- supplementation of metals into the media and periplasmic expression if metals are cofactors required for enzime stability;
best regards
Manuele
As mentioned by Adam B Shapiro and Manuele Martinelli low temperature expression should be tested; especially if you see that a minor part of the expressed protein is in the soluble fraction.
If you go for the fusion strategy be aware that getting a soluble fusion protein is not a guaranty that your protein is correctly folded and active. I have seen several examples of MBP making quite large aggregates soluble.
In some cases the protein in inclusion bodies is (almost) correctly folded and you don't need to denature it. Try resuspending the washed inclusion bodies in buffer with 2 M Urea, freeze it and thaw again. That can solubilize some proteins.
There is off course also the option of trying to refold the protein after purifying in the denatured state. There are several strategies so you will have to test to see which one works for each protein. Since your protein is his-tagged you can try refolding while it is bound to the column.
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