Dear All,
Please suggest any best method, protocols (Solubilization, Refolding, and elution buffers) for refolding of high cysteine rich proteins expressed in E.coli (IBs).
Currently I am working in a protein (25kD) having 9 cysteines. Our protein pI is 8.9 and evertime I took pH 8.0 of different buffers.
Thanks in advance
Best regards
Navdeep
Hi Navdeep,
In the case of disulphide bonded proteins, renaturation buffers must promote disulphide bond formation (oxidation). The most common methods used to promote oxidation during refolding are: air oxidation; the oxido shuffling system; the use of mixed disulphides; and oxidation of sulphonated proteins. It's better to use oxido-shuffling reagents which allow for both formation and reshuffling of disulphide bonds. The most common oxido shuffling reagents are reduced and oxidized glutathione (GSH/GSSG). Typically a 1-3 mM reduced thiol and a 10:1 to 5:1 ratio of reduced to oxidized thiol are used to promote proper disulphide bonding.
Does the cysteines in your protein exist as disulphide bonds or as reduced forms ?
Hi Navpeev
i do not would be pessimistic but refold a protein with 9 cysteines is quite hard.
The only recommendation that i can provide to you is perform it at high diluton to reduce the probability of aspecific intermolecular S-S bond formation which may lead to aggregation.
Probably the fastest way to try is test direct redolding in a panel of buffers containing different GSH and GSSG ratios and check the results not only monitoring the protein precipitation but also loading the non precipitate samples in SDS page (reducing and non reducing) to see if non-reducing condition you have the right MW or you have aggregates that are not good!!
However i think that in parallel is better if you try to obtain it as soluble protein using strategies that allow to produce S-S bonds during the expression which are:
- periplasmic expression
- origami strain
Other organisms (eg mammalian cells)
good luck
Manuele
I realize that with 9 Cys residues, this protein probably has several disulfide bonds, in which case you should heed the advice given above. However, if this protein does not contain any disulfide bonds, then you just need to maintain the Cys residues in a reduced state during the refolding process, such as by including TCEP, DTT, or reduced glutathione.
Thank you very much Manuele Sir and Adam Sir for your valuable suggestions.
As you mentioned in your comment regarding run the samples in non-denaturation conditions, we face aggregation problems. In reducing gel we got bands at right position (25kD) but while analysing it using non-denaturation conditions proteins get stuck (aggregate) at the loading wells (250kD). I think proteins got precipitated not properly refolded. We had already use different GSH and GSSG ratios (1:5 and 1:10) and DTT in previous experiments.
With Regards
Navdeep
Thank you all for enlightening me with your exceptional contributions. Let me apply and i will update you the outcome.
Thanks again
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Column