I am trying to purify this eukaryotic protein (mol wt 60 kDa) in Rosetta strain. It is getting expressed in good quantity. However, there is another stubborn host protein (mol wt around 70 kDa) which comes along with it after IMAC. Have tried everything to remove this (size exclusion, ion exchange, HIC, Chaperone wash, high salt wash, Urea wash) and it is still there. Is there any way to remove this protein? Please help.
A possibility is that it is the same protein that is longer because of read-through of the stop codon until a second stop codon was reached. You could check this by doing a bit of sequencing.
Adam B Shapiro Thank you for the suggestion but I have already checked the clone and also the protein through western which shows band at 60 kDa.
Heparin can get rid of the chaperones. There are some inputs, particularly for HSP-heparin complexation affinity. If you can get an immobilized heparin, before or after the IMAC treatment, you may test the removal efficiency.
Secondly, you can elute the target from the gel. Using the gel matrix as a purification tool. There are passive or active elution strategies for the protein back extraction from the PAGE gel. In addition, prep cell (BioRad) is tailored to do gel-based purification. It would be hard to refold and denatured form would be in hand indeed.
Good luck with your experiment...
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