Hi all,
I have got a protein, expressed in P. pastoris vector, more than 97% (SDS-page) pure in the culture medium after cells separation. Despite the protein expressed has a his-tag, Nichel based affinity chromatography purification failed (the native protein does not bind to the resin).
Since the protein is almost pure I tried to concentrate and wash with a clean buffer using centrifuge concentration tubes. However, together with the protein other components of the medium got concentrated as well (probably some other macromolecule, bigger than 30 kDa).
My question is, what other methods would you recommend to "wash" the protein which will allow me to discard those medium components?
Thanks in advance for your help
Davide
First step affinity chromatography like IMAC rarely result in pure proteins, thus your result is normal and to be expected. If the sizes of the target and contaminant proteins are clearly different, then SEC may be a method of choice. However, any other chromatographic step (IEC, HA, HIC...) may also be useful. Since most of the contaminants have already been removed, small columns with high-resolution media can be used. https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-strategies-for-protein-purification.pdf may be of interest.
Thanks for your answer! Likely i do not have any other proteins in my protein samples. Judging by SDS-page my protein sample is pure but it contains other components present in the culture broth. In order to remove those i tried to wash with a clean buffer and reconcentrate using concentration tubes. However, during these washes it seems I'm not able to remove them. The protein (glucose oxidase) seems to aggregate under the form of dark brown clumps (glucose oxidase should be yellow), so i suppose there is some other high molecular weight compound still present in the sample. Also, do you think the brown colour could be simply attributed to the high concentration?
Dear Davide..
I'm not an expert of pichia pastoris but is it possibile that the culture media contain some molecole that affect the abikity of the protein to Bond to the resin as happen in dome cases with mammalian cells?
Because in this case tiy van try to perform a sort of partial buffer exchange before imac or by dialisys or performing 1 or 2 cicle of concentration/diluitons using an inac binding buffer.
In case that this approach failure, you can filter the surnatant at 0.22um, concentrate in up to small volumes with centrifuga concentrator, amicon or tff ( if you have big volumes) and rune a SEC purification step which will allow to remove impurities abd perform buffer exchange at the same time.
Good luck
Manuele
If you are using standard Pichia media, there shouldn't be large protein components from the media. I express single domain antibodies in Pichia and it is quite pure as media alone. There is histidine in your media and that will compete for the Ni-NTA resin, so you need to buffer exchange. I use tangential flow filtration to both concentrate and buffer exchange and then continue on to IMAC enrichment followed by SEC polishing for >95% purity.
You can see examples of the purity in fig 6 of this recent paper:
https://www.nature.com/articles/s41598-020-79036-0
Thanks to all of you for the help,
I am still a novice in protein expression, and I did not consider at all the presence of histidine in the medium. But this would be likely the reason why the His-tagged protein did not bind on the Ni-NTA resin. However, I won't have tangential flow filtration apparatus so I think I will concentrate and buffer exchange using centrifugation tubes (30 kDa cut-off).
Thanks again, and Thomas J Esparza congrats for your work on "nanobodies"
Davide
How about doing an ammonium sulfate precipitation? This might help to get rid of the contaminants in the medium.
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