I am co-expressing my recombinant protein cloned in pET28a along with chaperone plasmid pKJE7 which helps in folding otherwise protein goes into inclusion bodies. But during purification by Ni-NTA affinity chromatoghaphy chaperone protein co-elutes with my recombinant protein. I have done the gel-filtration chromatography of the eluted fraction which suggests that chaperone is binding with my protein. I have tried different strategies like eluting Ni-NTA bound protein with 10-600 mM KCl, increasing MgCl2(10mM), lowering the pH of the solution upto 6 and ATP/MgCl2 upto 10 mM but protein is still bound with the Ni-NTA along with chaperone. Kindly suggests what can be done to resolve the issue?
Chaperones bind their clients mostly through hydrophobic interactions. You can try to dirupt the complex by addition of glycerol (cca 10%), sucrose (up to 500 mM) or by detergents. This could be done during loading and washing Ni-NTA column or as an additional purification step using gel filtration column.
Chaperones bind their clients mostly through hydrophobic interactions. You can try to dirupt the complex by addition of glycerol (cca 10%), sucrose (up to 500 mM) or by detergents. This could be done during loading and washing Ni-NTA column or as an additional purification step using gel filtration column.
I agree with the comments of Petr and would like add one more. If your protein is stable at low pH (~ 4.5 - 5.0) your can make elution from Ni-NTA column at this pH and load the concentrated eluted fraction directly on a gel-filtration column equilibrated with a buffer with pH 5.0. Usually, at this pH all hydrophobic interactions are prevented and your proteins will dissociate.
Based on what you see from Gel Filteration peaks on SDS-PAGE, your protein of interest is complexes with the chaperone protein and it appears the binding is very strong.
you need to find out the nature of binding before you attempt to break it. Is it electrostatic, hydrophobic, or disulphide bridges (if possible).
What is the molecular weight of the contaminating protein? is it close or very different from your protein?
Looks like you have hydrophobic interactions and adding some detergent might help your case. Try adding 0.2 % lauroyl sarcosine or some other similar detergent below CMC to your protein samples and see if they still bind together using sizing column.
If they fall apart and their molecular weights are different enough to separate on sizing column, you can get it and dialyse out the detergent. if the molecular weights are close then you can start with Ni-NTA chromatography in presence of detergent.
Try to include DTT or TCEP in sizing chromatography if you suspect possible disulphide bridges.
Good Luck
Venu
I had similar problem and the protocol of Rohman and Harrison-Lavoie (Separation of copurifying GroEL from glutathione-S-transferase fusion proteins. Protein Expr. Purif. 2000, 20: 45-47) worked perfectly. Briefly, you can denature by heat lysate of uninduced bacteria and add it to your lysate of interest. You should simultanously add ATP and MgCl2 and incubate shortly in 37 C degrees. Chaperones should bind to denatured protein which was added in excess. Then you can continue with purification procedure.
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