I have successfully expressed a soluble fraction of an enzyme after having heat shock proteins co-expressed with it. Cells were broken by sonication and further centrifuged. The cell lysate was then subjected to NI-NTA column, and eluted with 250 mM immidazole. when i checked on SDS-PAGE, i found other proteins like total 9 bands including 5 heat shock proteins.
I tried to add 2 M urea during purification to eliminate those protein, and it did not work. Any suggestions ?
Hi there,
The risk with HSPs is that when getting rid of it your protein of interest might precipitate because not fully folded. You might provoke the release of HSP from your protein by adding ATP to your washing buffer to trigger HSP ADP/ATP exchange.
Those proteins do not have an aminoacid sequence particularly enriched in His residues? In any case, you could try immnoprecipitation using His tag antibodies following column separation to further purify and concentrate your protein.
Sometimes you get quite unspecific binding on Ni-NTA columns. Washing with small imidazole concentrations (something like 20 mM) can help. Or, do you have the possibility to perform an FPLC/HPLC afterwards?
Thank you guys for sharing your experience here.
Dr.Dominique, i guess i ll suggest it to my lab mate and will see what does she think !! So far i have no background about your idea. Thanks alot for sharing
Dr.Vitor Teixeira, the proteins have no His sequences, these proteins are for folding so i guess there are chances that proteins are still bonded to the enzyme so i couldn't get rid of them, maybe!!
Dr. Lukas Winter, I usually wash with 10 mM immidazole for like 2 or 3 washes, and i still get these results. That's why i suggest those proteins might be bonded to the enzyme it self, What do you think ?
Dr. Babak Andi, i guess i will consider your suggestions and will go through it by next week. Thanks alot
Hii
I think you might need to increase the number of wash.Also the concentration of aroung 20-25 mM imidazole can be used for washing.While eluting you can use a gradient of imidazole concentration say 100 mM to 500 mM and optimize.In some cases,100 % purity is not attained by Ni-NTA alone and so additional purification procedures like ion exchange may be required.
Use wash buffer as lysate buffer (20 mM sodium phosphate, 500 mM NaCl, 40 mM imidazole, pH 7.4)
Do gradient elution with 20 mM phosphate, 500 mM NaCl, 250 mM imidazole, pH 7.4)
Wash 20 CV with wash buffer.
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