I am trying to purify a protein involved in DNA repair and is a part of supra-molecular complex. This protein was over-expressed using pET28a construct in BL21 2DE3 cells but its induction was very less (almost NIL). I switched to codon optimized strain (Rosetta 2DE3) cells which gave me a decent expression but fairly large amount of protein was going in to insoluble fractions. so i used triton X 100 (0.1%) in lysis buffer but it did not make any difference. Finally, use of sarkosyl (0.5 to 1%) gave me very good recovery in the soluble fraction, but then i am not sure whether its use is going to affect any of down stream studies (biophysical and crystallization) I wish to carry out with the purified protein. Can somebody tell me the dos and donts for using sarkosyl in protein purification strategies.
Use sarkosyle solubilized recombinant protein for DNA repair simple assay. If your rprotein shows activity means your protein is properly folded at that concentration of sarkosyle. Gradually decrease sarkosyle cocentration and check the activity of your protein. Tolerable Sarkosyle concentration shall vary for different proteins.
Thank you for the suggestion Gopal sir, but this protein works in DNA repair pathway only when in assembly with other interacting partner. And this project involves to bio-physically and structurally characterize this protein first then carry out PPIN studies. I also agree the concentration of sarkosyl that might affect the function of a protein varies from protein to protein. So, all i wanted to be sure of whether sarkosyl amount is gonna affect the folding or any other structural aspect of my protein. has anybody faced such problems during their studies using sarkosyl?
You may co-express your proteins in E.coli host if you know the interacting partner. Co expression may reduce inclusion body formation. One partner may cover hydrophobic patch/portion of other protein and thus solubility increase. You can use pet Duet vector or two compatible vector for coexpression.
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