Contamination after purification - (Dec/09/2005 )
We have trying to purify a 70 kDa protein under native conditions however our protein is always co-eluting with an endogenous E. coli protein that we believe to be SlyD. It appears at around 20 kDa on SDS-PAGE. Our protein of interest has a 6X His tag followed by a SUMO tag in the pET SUMO vector (Invitrogen). I have tried several purification kits, including the Ni NTA resin from Invitrogen and Novagen, as well as the BD Talon Resin that uses a Cobalt based resin.
At this point we have only been eluting with high concentrations of imidazole (150 - 250 mM).
I was wondering if anyone has had similar problems and what they did to get rid of this contaminant. I have been able to get pure protein only after A LOT of washes and the yield is terrible.
If anyone knows has to resolve this problem or has some suggestions I would greatly appreciate it, thanks!
I will verify if this 20kDa protein also contains the His fusion tag. It can be verified by running the Western Blot and probe it with anti-his antibody.
Please check there is no 6xHis fusion tag in your gene of interest and count it from the START codon.