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.