Hello. I am a graduate student making recombinant proteins at university.
Recently, I have succeeded in the expression of recombinant proteins and purification and refolding using Ni-NTA, and I want to verify that the refolding is done properly.
How can I know if the recombinant protein has been properly refolded?
In addition, I performed SDS-PAGE of the recombinant protein that I purified, and one more protein was detected except for the target protein.
Can you tell me what this is supposed to be?
The other protein may be host protein (you may have overexpress using bacterial system) or degradation fragment of your protein. You can check the refolding status using CD or by activity assays.
Hi,
You could take a sample from the SDS-PAGE and sequence the protein in this contaminant.
Regarding checking the folded state, you could use 1H NMR to check for signals below 0.0, those signals indicate a proper folding state of proteins. When no signals are observed below this value, it means the protein is not folded.
There are some papers that explain this,
Best,
Ismael
Dear Min Cheol Kim
What do you mean for refolding using Ni-NTA? Loading with denaturant (eg urea or guanidine) and elution with out denaturant?
THe absence of precipitation and the stability of the protein under concentration with ultrafiltration is certanilly a good indication that protein is refolded.
However you can run other experiments to further characterize it:
eg:
- SEC --> Folded protein are generally monomeric or dimeric while unfolded protein tends to aggregate and in SEC show a HMW oligomeric forms
- DSF --> Folded proteins show a stong transition (melting temperatures) while unfolded protens show no transition and with an high basal fluorescence (se na exmaple at minutes 2.30 of the following video
https://www.blogger.com/video.g?token=AD6v5dzr9KqZslTx9NeEjXx1XyPti8dU15eqYoE9TT8OEDaZQcNINuDKwzCAzVXkFWZA0btipKTr81doABh-VjpprwCcRukVcLhAXE_T5BzPxYbeQQA7VMFmc4DdpjS4XbOfxkwAhIWu
. NMR --> Peack dispertion in 1H NMR spectra is indicarive of protein folding. Folded protein show good dispestion while unfolded protein show strong peaks localized all in the same region.
good luck
Manuele
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Column