I am looking for a way on how to increase the yield of my protein of interest while reducing the amount of contaminated protein together.
I am using Imidazole in this Native purification.
10mM is used in the Native binding buffer,
20mM is used in the Native Was Buffer, and
250mM is used in the Native Elution buffer.
However with this recipe I found my protein of interest is being eluted out together with contaminating protein.
Is there another way to increase my purity of the protein while maintaining the yield of the protein by using the native purification method?
Few things that could help :
- Increasing the number of Histidines of your Tag from 6 to 8 or 10 can do the job.
- Having a specific protease cleavage site (Thrombin, TEV...) between His Tag and protein can allow you to make a two step purification giving often good results : do a classical IMAC, cleave with protease during dialysis (to get rid of excess Imidazole), then perform a reverse IMAC (kepping the flowthrough).
Hope this will help you
Another possibility would be to use NEB's NiCo(DE3) strains, which have been engineered to eliminate the most typical contaminants of IMAC runs. Never tried them, but I'm curious to hear reports from the trenches.
By the way, what is the purity of your starting homogenate? You cannot realistically expect a high purity eluate if the purity of your initial preparation is low. If so were the case, you could use something else (i.e. ion exchange, hydrophobic interactions) before Ni-NTA.
Besides the previous suggestions, you can further improve the purity of your protein by adding a size exclusion chromatography step (e.g. Superdex 75 or 200) after the IMAC runs.
The easiest thing to try first would be to include a washing step with high salt concentration 500-2000 mM NaCl). This avoids unspecific interactions of proteins with the resin. Most His-tagged proteins binds strong enough, so you can increase the imidazole concentration in your wash step to 50 mM.
The more laborious solution would be to clone the protein with a more specific tag, such as the Strep-Tag.
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