I was working on a project that involve refolding of bacterial expressed Immunoglobulin domain. I used the same refolding buffer as described in the literature. The refolding process went smoothly, but there is very low percentage of protein with intramolecular disulfide bond as judged by the migration of protein in the presence or absence of DTT. What can I do to increase intramolecular disulfide bond formation in the refolded protein? As I looked through the google search results, one paper described the use of 2,2′-Dithiodipyridine to enable disulfide bond formed in synthesized peptide, I don't know if it works on protein as well?
Dear Ming-Chung
at which pH did you perfom the refolding?
Disulfide bond formation requireds alkaline (pH=8 i think is the best compromise), so in case you did the incubation at acidic pH you can try to repeat it increasing the pH.
I think that to try to improve the S-S formation you can try to leave the sample open under stirring or air influx (low flow). However, you can also try to use different ration of (GSH/GSSG) and/or cysteine/cystine pair.
Of course in case your proteins contains more than 2 cysteines, is it better if you perform the refolding/oxidation at high dilutions to minimize the protein-protein interaction and try to avoid aspecific intermolecular S-S formation.
good luck
Manuele
Well it's like the Anfinsen expt again
1)check that nothing in ur soln breaks disulfide bonds
2) 2,2′-Dithiodipyridine will of course help form S-S bonds than just cysteine bcoz every time 2 S-S bond are formed aromaticity extended from 1 ring to 3 rings and facilitating electron delocalisation so much will of course help
3) Keep the soln at room temp, exposed to air, give time, keep the pH slighly above neutral , use a universal indicator paper to check its more than 7 but not too alkaline around 8. If u use the 2,2'DTP ur rxn will be over fast, so measure quickly
Dear Ming-Chung,
Low efficiency is a frequent problem in protein refolding. We've refolded dozens of immunoreceptors, cell surface antigens, including immunoglobulins. Optimal conditions for different proteins can vary significantly. There are different ways to refold proteins: (1) directly add urea or GuHCl solubilized inclusion body of your protein into the refolding buffer, which typically contains arginine to prevent protein aggregation. One will then concentrate and purify the refolded protein; (2) using dialyzing bags to step-wise exchange refolding buffers, starting from high conc of urea to low, such as 8M, 4M, 2M, 1M, 0.25M, 0M urea buffers. During the key intermediate steps of 2M, 1M urea steps, arginine is added to help to enhance refolding yield. In this popular method, redox pairs are usually further added, such as GSH/GSSG, cysteamine/cystamine, cysteine/cystine etc to increase yield. One will have find out empirically their optimal concentrations. (3) there is on-column refolding approach if your protein is his-tagged. While in principle, this approach is similar with that of the step-wise dialysis method, this method is rapid. One caveat is that the redox should be compatible with the Ni-NTA - low concentrations of many common redox pairs are compatible. You can check out details one of our method paper where we successfully refolded multiple immunoproteins:
Zhai L‡, Wu L‡, Li F, Burnham R, Pizarro JC, Xu B.A rapid method for refolding cell surface receptors and ligands. Sci. Rep. 2016, 6, 26482.
Not every protein can be refolded, although I know many immunoglobulins can be readily refolded. One quick you may check is if your protein has free cysteine, if it has, this extra free cysteine can be problematic - it can lead to incorrect disulfide bonds therefore it will decrease or even block the refolding.
You mentioned that you expect to a shift with and without DTT treatment in SDS-PAGE. This is often the case but occasionally a protein is refolded but there is no such shift. A sharp size exclusion peak may be a better read-out to quickly determine if a protein is refolded. Of course, one will have to test using additional biochemical and biophysical and functional tests for sure a protein is refolded as the native form and is functionally active.
Best of luck.
Hi Andrey Grishin
,I am interested in conjugating two of my proteins via S-S bond. Could you please share more details about how to conjugate two proteins by S-S bond? such as pH condition, temperature, addition of agents if necessary...
Thanks!
Thanks Andrey Grishin
!You were right. My case is more complicated. My proteins are folded and each have 5 cysteines and 4 of them form 2 S-S bonds in each subunit. I'd like to conjugate the 5th cysteine in each subunit together.
Yes, they are already folded. Dialysis at pH 8 to promote ss bond sounds good. We don’t have CuCl2 so I am planning to conjugate them by dialyzing against an redox buffer such as addition of reduced/oxidized gluthatione