I have a problem with purification of phosphorylation and nucleotide-binding domains of ATPase (P/N domain). I cloned DNA seq. into pET28a (N- his tag, 32kDa ) and transformed into BL21 Gold. The problem is that I have two degraded bands (at ~ 30 and 27 kDa) and DnaK band. I removed Dnak by Mg-ATP, but I cannot avoid the other degradation bands; however, I used 5X protease inhibitor cocktail (sigma), protease inhibitor cocktail Roch and 1 mM PMSF.
I also tested urea-treated lysis buffer and I got a very faint band of only 30 kDa and strong band of P/N domain. How can I solve this problem?
Other questions:
1- Is the presence of DnaK sign of mis-folding or instability of my protein? And can this cause degradation of protein?
2- Is the presence of protein in phosphorylated state causing instability of protein or attracting certain protease? And can the phosphorylated state of protein cause isoforms?
DnaK recognize extended peptide stretches with some specific preferences in sequence. Dnak bound to the fusion protein is an indication that you have accessible peptide unfolded regions, that may belong to your protein or to the fusion protein (it is probable the peptide junction between the fusion and your protein if you are using a fusion protein approach). Some specific protease recognition sites (i.e thrombin) are prone to strongly bound DnaK.
However, is your protein properly folded? You sould probably need to check this first. DnaK in addition to proteolysis during E. coli protein expression is a good indication of a miss-folded / unfolded protein.
I cannot answer question 2.
Thank you Eduardo. I tested another vector "pProExhta" with trc promoter and Tev protease site, the degradation level decreased but the chaperone DnaK expression was high . I tried to remove it with 5mM MgATP and 20% glycerol in washing buffer but still co-purified. but the question now what is th effective approach to remove the DnaK? by the ion exchange?
btw, the lysis buffer is 50mM tris-SO4, 300mM NaCl, 4% sucrose, 20% glycerol, 3mM MgCl2 20mM imidazol.
thank you in advance
To improve DnaK removal we included unfolded E. coli proteins in the washing buffer. In this way the exchange of Dnak between your protein and the E. coli proteins favor the chaperone removal. Take a glance to our paper: http://www.sciencedirect.com/science/article/pii/S1046592802000244. or send me your email address and a send you back a copy of this work.
Eduardo
Thank you again Eduardo. I know your paper, it is very interesting. but i remembered that the ATP washing with or without unfolded proteins causes removing of DnaK, is it right?
anyway, I will try this too. another question please, do you think addition of detergents like Titron x-100 can enhance the removal of dnaK?
thank you
Dear Ahmed
I quote here some paragraphs of the discussion of our paper “… in our hands, addition of ATP to complex mixtures as bacterial lysates containing fusion protein does not reduce DnaK contamination and usually increases it.” “When denatured proteins from E. coli, which serve as substrates for DnaK, were added to the washing buffer a significant enhancement of DnaK removal by MgATP was clearly observed”. This answer your question. We use this protocol usually in our lab and we have observed this repeatedly with different proteins.
Concerning Triton, in my opinion the detergent will not improve the release of DnaK unless you have some membrane contamination. In this case you should detect other protein bands in SDS-gels. In some cases, we have observed that ultracentrifugation of the sample prior affinity purification improve the quality of the target protein, probably by removing small membrane fragments and unfolded proteins.
Best regards,
Eduardo
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