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Nonspecific proteins eluting with HIS-tagged peptide


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#1 Ahrenhase

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Posted 03 May 2016 - 05:39 PM

I know this topic has been discussed ad nauseam; however, I was interested to hear if the responses change considering my unique circumstances.

 

I am expressing a ~12kDa recombinant protein in CHO cells.  I designed the construct so that it is secreted into the supernatent and the encoded protein has a C-terminus 6xHIS tag.  After 5 days, I collected supernatent by centrifugation and dialyzed in 50mM sodium phosphate, 300mM NaCl, and 10mM imidazole.  I then "purified" my protein on a cobalt column (FPLC), eluting with a gradient of imidazole; I found my protein of interest elutes at ~35mM imidazole.  Unfortunately, I'm also co-purifying a lot of high molecular weight (> 40 kDa) non-specific protein.  Given that untransfected CHO supernatent does not contain many cobalt-binding proteins, I believe that these nonspecific proteins are binding to my recombinant protein, given the high hydrophobicity of my recombinant protein.  My question is how can I acquire a more purified 12 kDa recombinant protein.

 

Here's what makes my circumstance different from most: I don't care about how denatured my protein becomes since functionality is not something I'm concerned with.  

 

What would you recommend?

 

A denaturing HIS purification using urea or guanidine?

 

Perform size exclusion chromatography on the cobalt purified sample I have now?  If so, what size stationary phase should I use?

 

Switch to affinity chromatography?  If so, what's the most cost-effect method and can the affinity tag be cleaved off?

 

I'm out of my element with this stuff; any help is greatly appreciated.  

 

 



#2 mdfenko

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Posted 04 May 2016 - 05:16 AM

you could try increasing the salt concentration to defeat protein-protein interaction.

 

denaturing with urea may do the trick but bear in mind that if the contaminating protein is actually binding to the matrix (due to exposed his residues), rather than your protein, then it may still bind.

 

sec on superdex-75 (or equivalent) may work to separate the proteins. superdex peptide may be better if you don't care if the contaminant(s) elute in the void volume.

 

his-cobalt (or nickel) is affinity chromatography. do you mean a more specific affinity matrix (like for your specific protein)?

 

affinity tags can be cleaved off easily if a specific cleave site is also incorporated in the construct.


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#3 labtastic

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Posted 06 May 2016 - 04:00 PM

What concerns me the most is why your his-tagged protein elutes with 35mM imidazole. That seems very low, even for cobalt resin.

 

How certain are you that you're purifying what you think you're purifying?


Edited by labtastic, 06 May 2016 - 04:03 PM.


#4 Ahrenhase

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Posted 28 May 2016 - 11:34 AM

What concerns me the most is why your his-tagged protein elutes with 35mM imidazole. That seems very low, even for cobalt resin.

 

How certain are you that you're purifying what you think you're purifying?

I just know I have a low molecular weight protein in my transfected sample that represents the correct molecular weight that is not in my untransfected sample.  Unfortunately, the HIS epitope seems to be concealed during western blot analysis so I can't get confirmation (unless of course it's not actually there).  I'm just going to try a GST tag.  



#5 labtastic

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Posted 30 May 2016 - 10:01 AM

Well if you are purifying the protein on cobalt resin then in principle it can't be all that concealed, at least when it is natured.

 

I agree a different tag might be the way to go. Consider an Ntermainl his tag too (if you can still get it excreted).  Cleavable superfolderGFP tags can also be helpful.






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