His tag protein purification problem - (Jun/12/2004 )
hi all,
I work with a his tagged protein (~43KDa). The protein has absolutely no solubility problem after overexpression from a pET15b in pLys S cells. The protein does not bind to Ni-NTA. It does not bind to anyother column also. But upon denaturation by 8M urea it binds to a certain extent in Ni-NTA. Almost always the protein goes in flowthrough in any column you put. I tried Ni-NTA, CM-Sepharose, DEAE with various pH ranges (8, 8.8, and pH 6.4 for CM Sepharose as well since the proteins theoretical pI is 10.02). I confirmed it by doing a wester blot with anti-his anti bodies in all cases. Can amy body help??
hi, I'm 2nd year phd student working on membrane proteins and I have an msc in enzymology
I have worked with tagged proteins but never with his;
43kDa is the size of your protein including the tag? If so, did you take into account the extra amino acids when you calculated the theoretical pI ?
did you pH the buffers very carefully? do not follow Maniatis' protocols of mixing powders at some ratios, as the resulting pH can easily be 0.2-0.5 different and it DOES make quite a difference in affinity work.
did you try to bind your protein to the Ni column in the cold room? lower temperature in many cases assists binding!
did you have DTT in all your solutions your protein was in? This includes lysis buffers, dialysis buffers etc. 1mM DTT will prevent the majority of missfolding events.
Good luck
Your first objective would be to track the protein size using a Sephacryl S-500 gel filtration and check if the target entity exists as a dimer/trimer/teramer or multiples thereof. Inclusive tight aggregates prevent binding of proteins to columns. RNA polymerase core enzyme comprising of alpha and the beta/beta-prime subunits with a piece of embedded DNA does not bind to any column despite presence of denaturants. Could your target entity be a DNA or RNA binding protein?
43kDa is the size of your protein including the tag? If so, did you take into account the extra amino acids when you calculated the theoretical pI ?
---Yes, Its including the Tag
did you pH the buffers very carefully? do not follow Maniatis' protocols of mixing powders at some ratios, as the resulting pH can easily be 0.2-0.5 different and it DOES make quite a difference in affinity work.
did you try to bind your protein to the Ni column in the cold room? lower temperature in many cases assists binding!
Yes, i dont follow the mixing powders...i adjusted pH by HCl Concentrated for Tris. to pH 8.0
did you try to bind your protein to the Ni column in the cold room? lower temperature in many cases assists binding!
Yes i did...no use..
Did you have DTT in all your solutions your protein was in? This includes lysis buffers, dialysis buffers etc. 1mM DTT will prevent the majority of missfolding events.
No i didnt ..will try defenitely
Your first objective would be to track the protein size using a Sephacryl S-500 gel filtration and check if the target entity exists as a dimer/trimer/teramer or multiples thereof. Inclusive tight aggregates prevent binding of proteins to columns. RNA polymerase core enzyme comprising of alpha and the beta/beta-prime subunits with a piece of embedded DNA does not bind to any column despite presence of denaturants. Could your target entity be a DNA or RNA binding protein?
Yes..i tried putting on Sephadex G75 and Sephadex G200 from Amersham on Akta FPLC..all the cases the protein came in void volume.
Its supposed to be a DNA cutting and rejoining enzyme (integrase) but its site specific.
I'm surprised that your protein stuck to the column at 8M urea... I had the impression that urea (as well as triton) prevents ANY binding on affinity columns (could somebody please correct me if I'm wrong).
Also, could it be that post translational modification plays a role in the lack of binding?
I would certainly try to express the protein at room temp (22-30'C) and try the binding again. What buffer do you resuspend the cell pellet in? How much NaCl do you have in your buffer?
Most likely, the His-tag is embedded in the protein and therefore only exposed when completley denatured by 8M urea. Could you put the His-tag in the other end of the protein?!
Alternatively, you could purify it denatured (8M Urea) and then renature it afterwards.
good luck--
Also, could it be that post translational modification plays a role in the lack of binding?
I would certainly try to express the protein at room temp (22-30'C) and try the binding again. What buffer do you resuspend the cell pellet in? How much NaCl do you have in your buffer?
The Qiagen catalogue says Affinity binding (particularly with Ni NTA) is UNAFFECTED by conditions like 6M Gu-HCl or 8M Urea.
I dissolve the protein in Tris HCl buffer (25 mM) pH 8.0. I initially had 300mM NaCl as there was no binding i reduced the NaCl concentration step by step and i even tried with buffer with no NaCl at all.
I used MgSO4 and DNase along with 25mM Tris.pH 8.0 but the protein precipitated and went in to pellet. But when i shifted to use MgCl2 instead it remained in Supernatant.
Presently i use 25mM Tris.Cl pH 8.0, 10mM MgCl2, 1%NaN3 and 20ug/ml DNase.and protease inhibitor cocktail from SIGMA.
I suppose there is a formation of soluble aggregates that prevents the His Tag from getting exposed. I tried with detergents like Zwittergent (0.1%) most of it comes in the flowthrough again.
I grow the cells at 25 C for 8Hrs. The expression is huge.
Is there any way to break the soluble aggregates other than denaturing...because, i want my protein to be a homogenous population and refolding may yield populations which are not homogenous even though they show activity.
I dont want to put the Tag on the other side as well becuase, the active site residues are close to the C-terminus of the protein and it might defenitely afect the activity.
Sincerely,
sankar
Hmmm, sounds like you've eliminated most things leaving just His-tag accessability as a problem, though it's doesn't add up that the protein is not bound well even under denaturing conditions.
Here are a few things to mees around with while you curse your project:
If you don't want to put the tag on the other end of the protein maybe putting on a poly-Gly linker to bring it out away from the rest of the protein. Might help if you think it is tucked away against the protein. May, however, hinder/alter protein activity too much.
If you think it is aggergation then go with MORE salt (up to 2 M NaCl or4 M MgCl2). Also additives like glycerol and EtOH can help prevent aggregation. What about detergents (tween, triton, NP-40 all up to 2%)?
If you do use DTT, NOT more that 1mM.
Is your protease inhibitor cocktail from SIGMA the His-tag version so it doesn't contain EDTA?
A lot of this is straight out of the Qiagen booklet so you may have tried it already.
Good luck!
I would DEFINITELY drop MgCl2 and DNAse (MgCl2 is added as a co-factor of the DNAse as you probably know). It has caused me problems before (but I work with DNA-binding proteins so it could be a very different matter).
If you want to get rid of the DNA, try PEI (polyethilenimin) precipitation (look it up on google).
You may wish to do an ion-exchange step prior to HisTag column. This weeds out interference that prevents His-binding to the Ni-NTA column. Expression is the more critical element of your exercise, not purification. One can always purify using a variety of methods out there.
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