Hi all,
I do the expression of wt unlabeled and fluorinated domains of euakaryotic proteins in E.Coli BL21(de3) cells. Over the past few months, I have been noticing that during our FPLC purifications, there is a bacterial protein of high molecular weight between 50-75 KDa which elutes before our His6-tagged proteins. Moreover, in our failed expressions, the yield of the bacterial protein is a lot suggesting the bacteria is directing its resources towards the synthesis of the bacterial protein.
Also, in some cases, we have been able to get the unlabeled protein but not the fluorinated one which is expressed in defined media (mixture of amino acids, salts and aromatic amino acid). In other case, protein expression failed in both the cases for both the wt and fluorinated one. That's why, I can't understand if the cell strain is important or if I have to vary the expression conditions depending upon the protein in question.
Please provide any inputs, if you have observed this behavior before.
Thanks.
Hi Sebastian,
Thanks for your reply. I think that purification and separation of the bacterial protein from my desired protein is not a problem. I just feel that the yield of the bacterial protein is greatly enhanced when the expression of the desired protein is failed. This suggests to me as you have pointed out, that the protein may require different expression conditions. I didn't analyze the pellet which I should do.
Do you have a protocol for analyzing the pellet? Because it is so concentrated, generally I have the problems of smearing of bands and it is difficult to identify isolated bands.
Hi there,
According to the range of MW you give the contaminant is ArnA. It gets highly enriched during NiNTA if the level of expression of your POI is decreased because both are simply competing for binding to NiNTA.
Hi Prakriti,
1. As mentioned above, because the Ni-NTA resin may bind some proteins non-specifically, it is normal to have many protein bands only after affinity chromatography. You need to purify the target protein through further operations, such as gel filtration or ion exchange;
2. If these bands are the degradation bands of your target protein, then suggested that the conditions of expression and purification are not sufficient to ensure that your protein is in a relatively stable state, and it is recommended to optimize the conditions, e.g. try more constructions and mammalian cell expression systems…;
3. If these bands are endogenous and of high concentration, it is recommended that you optimize the induction conditions or try other expression systems.
Hope it helps!
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