His-tag protein purificaton, cotamination with 40-43kD protein - (Jul/27/2005 )

Hi there,

I'm trying to purify the recombinant protein.

My recombinant protein is not actually 6xhis tagged. I use pThiohis B vector (Invitrogen) for producing the recombinant protein and my interest protein has fusion part which should have high affinity to Ni-NTA because of its conformation (it has only 3xhis though).

Anyway, I'm now trying to purify that recombinat protein using Ni charged affinity resin (Probond: Invitorogen) and I could get my recombinant protein partly purified.
However I got very high amount of one contaminant protein at the same time.
It is about 40-43kD and contaminated with very high amount.

Does anyone know what is this protein and how to remove this?
I'm using E.coli TOP10 from Invitrogen which is similar strain to DH10B
Does this contaminate protein come from only TOP10 strain?

I would be grateful for any suggestions.

Thanks

Any chance it's an incompletetly transcribed or partially degraded protein of the same type? Some recombinant proteins will get partially digested by cellular proteases before you have the chance to purify it. Also, if there's a string of somewhat rare codons that your strain is deficient in, you could be getting partial transcription. You didn't tell us what size your intended construct is, though - these two possibilities are only feasible if your construct is bigger than the contaminant.

If it does happen to be one of these two, you could solve the first by including protease inhibitor cocktails during lysis, growing at lower temperature, or using a different strain for expression. To solve the second would require the use of a codon+ strain like Rosetta.

If it's a true contaminant, though, and not just a stable fragment of your intended construct, you can probably get rid of it by ion exchange or size exclusion chromatography. It's one extra step, but that's better than trying to incorporate an unknown contaminant into any subsequent data analysis.