All three recombinant proteins varying in sizes (17 kDa, 26, Kda and 29 kDa) are co-purifying with a 55 kDa protein. Two proteins were extracted from E.coli [whole cells pellet] with Buffer A (50 mM Tris-HCl [pH8.0], 8M Urea, 250 mM NaCl, 0.5 mM EDTA and 5 mM b-ME). One protein was extracted from inclusion bodies using the same buffer A. This has happened twice in a row with these three proteins and I have no clue about it. Contaminant 55 kDa protein is not a dimer or trimer of any of these proteins as the position of this band on SDS-PAGE is same in case of all three proteins.
After binding proteins to the Ni-Sepharose, I'm washing with buffer A (containing 20 mM Imidazole and 1M NaCl) and before elution 5 column volume of buffer A. Elution buffer is also buffer A with 250 mM Imidazole.
I have same batch of competent BL21(DE3) that I have been using for the last few months and I have used for many other proteins without this new problem.
Hi Abdur
I've intermittently observed a contaminating protein ~55 kDa, after prepping protein using Gdn. It's hard to say if it's the same as what you're observing, but the contaminating band was always very minor and was removed by anion exchange on a HiTrapQ (quaternary amine) column.
If this is not a minor band we're talking about, one major difference between my purification scheme and yours is that I renature my protein on the column. After washing with the lysis buffer, I run buffer without denaturant for about 5 column volumes, then elute with native buffer plus anywhere from 200 to 500 mM imidazole (construct dependent). I don't know how this would make a difference, but perhaps the contaminant binds Ni-NTA more strongly in its native state or perhaps it aggregates.
I hope this helps,
Jordan
Hi Jordan,
Thank you for your input. The intensity of 55 kDa bend is almost same as the target recombinant protein in all three preps for three different proteins. Two preps were done at the same time.
I have found contaminating 55 kDa proteins that come from the large subunit of antibodies and, in some cases, from keratins present in the air. I do not know all the specifics of your work or your environment but I have dealt with these issues before in protein purification. I hope this helps.
Hi Gustavo,
Thank you. I know for sure that its not heavy chain of antibody, however, Keratin could be a possibility. So when you had this problem, how did you solve it?
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