Hi,

I guess there could be additional surface-exposed histidines in your protein that can be coordinated by the column material you're using for your His-tag purification.

Hi there,

If a protein binds to NiNTA and is totally eluted by 300mM imidazole you can't say the interaction is not specific, on the contrary it is specific. The main issue is are you recovering the full amount of protein loaded onto the gel? Second issue: can you spot the difference on SDS PAGE or SEC between cleaved and intact species and/or formally identify free tag on gel ? Third issue : is the intact protein also eluted with 300mM imidazole?

My opiniion is either the cleavage is unefficient (but obviously you can spot the tag alone) and your are eluting intact protein or the tag remains tightly bound (but non covalently) to the rest of the protein.

Hi ,@Eef Dirksen

Thanks for your answer.

I checked the sequence of my protein . please look at the attachement.

In fact, not many Histidine in my protein (3, just 1.1% in all) .

I don't know whether the Arg (corresponding residue to His) can be also coordinated by NI-NTA using in my purification.

Frankly ,there are 18 Arg(s) in my protein .Its amount is substantial .

Hi ,Dominique Liger

Thank you for your kind heart .

Yep,that 's  nice of your issues.

The efficience of cleavage ia at least 90%  which could be found in SDS-PAGE gel . I really got all cleaved protein from 300 mM imidazole elution  . After this strange phenomenon ,I elute my intact protein directly from column by 300mM imidazole ,and then process the 3C digestion in solution .

I quite agree with your opinion on the tag remains tightly bound (but non covalently) to the rest of the protein .Maybe that's right! 

so ,perhaps I will purify my protein utilizing GST -tag combinant .In this case may I could using protease cleavage in-situ...

Are you including imidazole in your wash buffer, and what salt concentration? The most likely explanation is that you have charged surface regions that are interacting with the nickel. 20-50mM imidazole should be enough to disrupt these interactions (I typically use 30mM imidazole). You could alternatively use increasing concentrations of imidazole to elute and look to see at what concentration your protein elutes separately from the his-tag.

I would normally IMAC purify my protein, buffer exchange to a low-imidazole buffer, cleave the tag and then load everything back onto the nickel column. Wash a few times with ~30mM imidazole and normally that's enough to dissociate non-His tagged proteins from the column!

Hi John

Yes , I wash my 3ml nickle column using 150ml wash buffer containing 20mM imidazole.

I agree with your opinion on that my protein have charged surface regions that are interacting with the nickel ...

I am now changing my protocol to purify this protein .

John,Thanks for your advice you finally proposed .It's a nice reference for my future purfication .

Yes, primary amines such as R and K can be electron donating to the outer electron orbitals and cause some reduction of Ni at the appropriate pH.  Glycine and glutamine can do this as free amino acids.  The amino acid composition shows ~13% basic primary amine residues but also ~8% carboxylic acid residues that could salt bridge with amines.  The positions of the 3 histidines in the sequence is relevant even though they constitute ~1% of the amino acids.  If their surface positions are contiguous or juxtaposed through the protein's  secondary and tertiary structure then they could confer significant binding to Ni;  human IgG's are an example of this.  Does it really require a minimum of 300 mM imidazole to elute the de-tagged protein from Ni?  What would happen in the presence of 4 M urea used to dissociate any bound components and change the conformation as well?  Does elution occur at acid pH where the tau nitrogen of His gets protonated as well as charged groups?

hello,@Grant Shimamoto

Thanks for your reply.

your answer  makes me clear thant before.

I don't know whether its elution really require a minimun of 300 mM imidazole .Because I used 300 mM imidazole to elute it following a 20~30mM imidazole washing process. Maybe 100mM can also elute it...I don't konw...

with regard to what you said :What would happen in the presence of 4 M urea used to dissociate any bound components and change the conformation as well?  Does elution occur at acid pH where the tau nitrogen of His gets protonated as well as charged groups?

because I need a soluble sample to crystallize.,you know crystallization requires a high quality of homogeneity sample .I think the sample eluted by urea or acid pH is misfit crystallisation  . so I have not eluted it by urea or change pH .Maybe it will be a interesting thing. 

Hi Dali CY Hu;

I agree with Dominique above but would add the following:

 1. When performing SEC, your Hist-Tag will appear right in front (small A260) of your salt peak (Conductivity peak) at the end of your run. 

2.  Your gel may not have enough resolution to see that small of a shift - I'd suggest NuPage Gels (Thermo-Fisher now) for the highest resolution - the MES or MOPS buffer systems helps resolves peptides in different size ranges - creates very tight banding patterns compared to Tris-Glycine.

3.  If High Arginine content is a root cause to binding, that would be very interesting.   That may have been reported by someone - because there is a possibility that high lysine might have a similar interaction.

4.  I have worked with nucleoproteins (pI ~10) from viral origins and I have not seen this behavior - Yet I had not looked for this because I was not removing Hist-tag to crystallizing my protein.  I did run an Imidazole linear gradients to characterize my protein's binding/elution characteristics.  I believe this reflects the availability of your Hist-Tag to bind to your Ni-NTA and helped me.  Some His-tagged viral capsid proteins have had some very low binding characteristics - eluting near 100 mM Imidazole.  The nuclear protein I mentioned above eluted at ~190 mM Imidazole however, I set the process at 300 mM Imidazole.

5. What detergents or stabilizers do you use for crystallization of your protein.  Perhaps you use a kit to screen for crystallization.  I have enjoyed the results I have obtained by purifying from yeast.  The codon table (codon usage) is an eukaryotic codon table, no inclusion bodies (like prokaryotes), reasonable yields - that's if you have a human target protein.

6.  Yes - stay away from Urea if crystallizing -  

7.  I have worked a lot with IgG's - Ni-NTA is more of an esoteric interaction.

8.  I have used either a French press or a micro-fluidizer to create cell lysates for purification.

Hopefully I have helped

Dan

Hi,@Dan Weaver

Thank you very much.

Your expertises are reasonable and infectious.I have some questions to consult you.

1.In fact,My SDS-PAGE's resolution could convince me.Please look at the attached figure.Our SDS-PAGE in TRIS glicine buffer can also distinguish molecules from approximate 10 amino acides difference.I know your kindness,but 6XHis have cleaved from my protein indeed at least from SDS-PAGE gel image.

2.Yes,Thanks for your reminding about the characteristics of Arginine and lysine.Maybe my protein shows so affinity to NI-NTA just because of the number of R&K&H  or N/Q/D/E.

3. No any detergent and additive in My protein.Do you think it is necessary to add some detergent to my protein to crystallize enve though my protein is not membrane protein?Did you have a case to crystallize by addition of detergent or additive in screening?What kind of detergent you have added?

4.As for your purification from yeast,this is your regular purification?Our purication system is seted up in E.coli because of its simpleness.Does yeast purification system can decrease the production of inclusion body?If it is ,it's a worthy doing.

5. Is Urea a absolute prohibition in crystallization?I have used 500mM Urea as a additive in my protein buffer to expect no aggregation of my protein.Though I have not got any crystal in this trays,the homegeneity is better than no urea sample's.I think Urea could abolish hydrogen bond,so some urea in low concentration could inhibit protein molecules from aggregating.If you have more experience in crystalization by additives to stablize proteins,I look forward to your sharing.

Thank you very much

Best regards,

Good Morning -

1.  That's a nice Gel - Bands are clear and very sharp and you are able to see the slight shift in mobility when the tag has been removed.  That is great.  I figured a 6 X hist-Tag would be more difficult to visualize - the relative size change looks ~ 5%.  My proteins ranged in the 70 - 90 Kd which would have represented 1.6% difference in size running in the upper range of the gel - little more difficult area to resolve smaller differences.

2.  Correct - right now you just don't know - which is ok.  Sometimes it is better to acknowledge that before embarking upon a series of experiments that lead you away from your goal - which looks like protein crystallization.

3.  Your intuition is correct to stay away from Urea and detergents - yet again I have not had the pleasure of crystallizing proteins.  I had an antigen/ms-antibody interactions which by function definition:

  a. Functional in an Elisa - non-detectable by western blot :  (2ndary/3tary structure necessary for binding of the antibody).  We elected yeast based upon that criteria - higher yields or higher specific activity antigen.  Problems we had were lower activity per mg, less full length protein.  Once again we avoided stuff like bug buster or commercial lysing reagents from off the shelf.  We learned early to use the French Press and to increase through put transfer to micro fluidizer.  Inclusion bodies are not a big problem when the antibody interacts strongly to your protein in a western blot.  However, they do hinderer when higher level structure is necessary for functional activity - Likewise -  crystallization represents a requirement for higher level of structure for functional activity.  I don't think Yeast will produce inclusion bodies - yet that may be a gap in my knowledge.  I'd like to know if they did? 

4. If no French press is available - they will be difficult to lysis chemically.  There are kits available, however, stick to the enzymatic ones and avoid the chaotrophic ones similar to bug buster.

5. Curious - what are your chaperones - I've used glycerol when I had too.

Best regards

Assuming your His tag is indeed cleaved, it is still possible for your protein to bind to a nickel column. You may want to look at the sequence to see if you have any His patches. But in any case your cleave protein should elute out at lower imidazole concentrations. 

You can run an imidazole gradient to see at what concentration your His-tagged protein elutes. Your cleaved protein should elute at a much lower concentration. If it doesn't I would suggest alternative methods to confirm you accomplished cleavage.

Good luck!

Hi,@Francis Lau

Thanks for your reply.

It's a good idea to elute my protein by somehow low concentraion imidazole thus separate cleaved protein from total system.

thanks more

Hi,Dan Weaver

Good morning.

Thanks for your opinions.

The problem about whether yeast would produce inclusion body may caused your misunderstanding.Theoretically,some proteins difficult to be foled would easily to form inclusion body if it be overexpressed in expessing cells.But if the expressing cell is advanced enough,which could decrease the content of inclusion body for the help of more folding factors.In this way,we can obtain higher yields. 

I have not utilized Yeast system to express proteins. I don't know whether yeast expression system is more more reliable than E.coli in reality.So I wanna ask you for some experience.After all,it is tough to transfer E.coli system to yeast for me.

BTW,the band of chaperones is a heat shock protein--Hsp70 able to help protein fold it make protein more soluble.

If your protein is correctly folded and you have urea in the elution buffer then can you remove the urea post IMAC, maybe by dialysis into stablizing buffer, then proceed with your crystallization?