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Problems with a nonspecific interaction on a Ni-NTA column

nonspecific Ni-NTA His-tag

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#1 jessiray189

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Posted 16 July 2014 - 09:24 AM

I'm trying to purify a kinase that is expressed with a His-tag. In order to keep the E. coli cells alive during the expression phase, we have to co-express the kinase with a phosphatase. Unfortunately, the phosphatase binds nonspecificially to the Ni-NTA column, and it binds with surprising strength. Normally, we run a Ni-NTA column followed by TEV cleavage and dialysis, then an anion exchange column, followed by a gel filtration column. Normally, a gel filtration column would solve the separation issue, but the other issue we have is that the kinase and the phosphatase are ~2 kDA apart in molecular weight, so it's extremely difficult to separate them. Does anyone have any advice about separating our kinase from our phosphatase? Thank you!



#2 labtastic

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Posted 16 July 2014 - 11:47 AM

I am assuming that the TEV cleavage is to remove the His tag on the kinase?

 

If so, then just run your TEV-cleaved kinase+phosphatase mixture back through some NiNTA resin. The TEV, cleaved his-tag and your phosphatase will stick to the resin while your kinase will flow through.



#3 jessiray189

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Posted 25 July 2014 - 05:59 AM

Thank you so much for your response! Unfortunately, I did do this, and somewhere along the line I lost most of my kinase. I'm starting to wonder if there isn't a problem with my Ni-NTA resin, along with the phosphotase sticking issue.



#4 labtastic

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Posted 05 August 2014 - 09:42 AM

I had a similar problem as you it sounds. I would express my proteins with a TEV cleavable c-terminal GFP-His6 tag. The TEV would cleave my proteins from the GFP beautifully, but I could not separate the my protein from the TEV afterward because my proteins either stuck to the column on the reverse Ni-NTA step, or the TEV would co-elute with my protein on gel-filtration even though they differed by about 130kDa. I know these same proteins when expressed without a cleavage GFP tag do not stick to NiNTA non-specfically, so it had to be something to do with the TEV site or the TEV itself (which was poorly prepped by a previous student in the lab).

 

I have since switched from using the TEV cut site to a Prescission Protease (aka Rhino virus 3c protease). I no longer have any of the problems I was having with the TEV-cleaved proteins. If I were you, I would consider modifying your plasmid by swapping out your TEV site for a prescission protease cut site, and of course use prescission protease instead of tev to cleave your protein.


Edited by labtastic, 05 August 2014 - 09:56 AM.






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