His-Trple A protein construct into DE3 cell, pellet were suspended in 10mM HEPES ph 7.4 250mMkcl and 10%glycerol, The resulting pellet resuspending in 100mM Tris ph7.0,0.5M=mM EDTA,5mMDTT,2 M urea, TritonX-100,
Then treated with extraction buffer (10mM HEPES,250mMkcl,6M guanidine HCl but after chromatography (NI-NTA affinity)and dialysis protein aggregated, what should i do
You check the pH of your protein and make sure that buffer pH is different from your proteins pI. Another thing that is possible that your protein is not folding. Making the hydrophobic residues exposed and protein collapse due to hydrophobic interaction. You can also increase the salt concentration. Before doing dialysis, you can go for size exclusion chromatography to check the protein quality. Hope this helps you.
Dear Sarita Subedi
from the composition of your buffer, it seems that your protein is expressed in the insoluble fraction and you need to exctrat it from IB using a denaturing agent as guanidine. Unfortunatelly is quite common the dialisis and removal of denaturing agent result in protein aggregation if it not succed in refold.
In my experience the best solution is try to optimize, protein domain, expression strain, host, conditions to obtain it directly in the soluble form and avoid the refolding step.
If this is not possible, you can try to screen a panel of refolding buffers changing the ph, ionic strenght, additives as arginine, addition of cofactors if present, to promote the protein refolding. But you need a lot of lucky.
good luck
best regards
Manuele
As Manuele said, if you can have a directly soluble protein, it could save you a lot of effort.
Now, you are trying to purify and refold a protein expressed as inclusion bodies.
there are a few things you could try to optimise.
1) check for protein purity after your Ni-NTA. (to load high-guanidine samples on gel, run the gel at 120V. make samples at a 10 or 20:1 loading buffer/sample ratio, spin down and take only the supernatant). If needed, for instance adding a low-imidazole rinsing step can significantly improve purity. Adding one more step of purification can also help (ion exchange, size exclusion, ...). Protein purity is very important!
2) with your pure protein, you can try to optimise the refolding procedure:
- protein concentration (usually ~1mg/ml, but you could try lower)
- pH of the buffer
- salt concentration/type (monovalent, divalent, K+ vs Na+)
- cofactors? type+concentration
- denaturing agent removal speed (-> volume of refolding buffer/sample, dialysis in 1 or 2 or 3 steps, in which case buffers can also have different denaturing agents concentrations, or even a gradient dialysis where you add low-denaturant buffer to a small amount of high denaturant buffer in which your dialysis membrane sits, while removing excess buffer)
cheers!
Hello Manuele Martinelli Ulric le Paige Aakanksha Singh Thank you so much, My protein sequence is 42 amino acid sequence and within this 9 cystein molecules. If the case of cystein molecule what should i do to prevent aggregation.
Thank you
ooh, short peptide with a lot of cysteines. Is it from some venom?
how many prolines do you have? if you have several, it could be worth it to make a slow refolding (-> dialyse to medium guanidine- or urea-containing buffers, to give time for the cis-trans isomerisation, and stepwise decrease denaturant until the end).
well, for cysteines, there are a few options then.
- if refolding: starting from the extraction part, I would strongly emphasize the use of copious amounts of fresh DTT or 2-ME (cheaper but a bit less efficient) in most buffers, all along, until the protein is well refolded. Maybe increasing the incubation time in guanidine at the beginning, and maybe even heating up a bit (careful, heating in guanidine, not in urea as urea degrades with heat and that can lead to lysine carbamylation). Using DTT only in the wash buffer is most likely far from enough.
- if trying to optimise expression of already-folded protein:
there are a few strains that have been optimised for S-S bond isomerisation, such as Shuffle and Origami strains (Careful, those grow slow!). Could be worth a try? and then you could purify your protein natively (fingers crossed).
anyways, keep in mind that no matter your efforts, some insoluble fraction will be unavoidable at the end of the refolding. what matters is yield and time-stability of the product.
Ulric le Paige , in sequence only one proline
You can contact me at the following email addresses:
- Badges
- Science topic
- Similar topics
- Organizational Psychology
- Training