Hi all.
I have a pure protein but in SDS-PAGE gel there is DNAk in the solution too ( It is together with my protein). In Superose6 column I have 3 proximate peaks which I cant separated. I think DNAk is together with my protein and It isn't finished the folding or the protein is badly folded. So I supposed the protein need end the folding but I dont know If I can do something in growth/expression E.coli step ( I use BL21(DE3) 0,5mM IPTG) or in protein purification, I use a typical buffer with 50mM Hepes ph:7,6, 200mM NaCl and 25mM Saccharose ( I proved other additives like L-Arginine, detergents.. and i didnt saw any effects) I sonicated the cells ( 2min 30%amplitude) and then I do HisTrap (Ni column)-MonoQ (anion exchange)-Superose6 (molecular exclusion).
Thank you
Perform in-column refolding when the protein is still attached to Ni(II)
Did you wash the fusion protein bound to the purification resin with MgATP ?
http://www.sciencedirect.com/science/article/pii/S1046592802000244
You must try an ATP/Mg wash in order to remove the DNAk. For the folding of your protein you must try to lower your temperature of expression (if not done yet). or test another E.coli strain for your expression.
Good luck
But In solution without DNAk could my protein fold? I expressed my protein at 20 Tº Overnight but I am going to prove to low the IPTG concentration from 0,3mM to 0,02mM IPTG.
something you can try is to coexpress your protein with chaperones using pGro vectors. Since the DNAk binds your protein during the expression, I will suggest that you start by inducing the DNAk to make sure that there is enough chaperon to help with your protein folding before you add IPTG. For the removal as I suggest before you can use the ATP/Mg wash.
Yes i try it. I am going to use BL21 pGroEL or pGroES for protein expression.
Thank you
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