Michael, It is an interesting coincidence! We have also the same problem a couple of years ago purifying chloroplast Hsp100 chaperones expressed in E. coli. ArnA is repressed by bivalent cations. So, try to express your protein using the E. coli BL21(DE3)pLysS (or any other similar strain) adding 10 mM MgCl2 to the culture medium during culture and protein expression. Adding MgCl2 solved the problems for us. Good luck!

Hi Michael,

we encountered a sililar problem with a large eukaryotic protein binding E. coli Hsp90. We tried ATP, ADP, a specific inhibitor of Hsp90 which promotes a conformational change, etc... Unfortunately, we did not succeed. I had the impression, that without the chaperon, our protein would just unfold again/fall apart, thus it was bound so tightly. You should anyway try ATP and ADP. Is there a known interaction partner of your protein? Could you co-express it, to stabilize your protein. Or is there a compound known which has the same effect? You coud try to disintegrate the complex by using chromatographic methods, which are coming out of fashion (in the days of affinity tags). Either you try ion exchange chromatography (IEX) or hydrophobic interaction chromatography (HIC), which both deal with physico-chemical properties. If cpn10/60 have a different pI than your protein, IEX could work. With HIC (where you add ammonium sulfate) you could achieve the same but based on the different hydrophobicity of the components of the complex. It is worth a try.

Good luck,

Magdalena

Hi Michael,

I have had similar chaperone contaminants when purifying myosin V from insert cells. I found that IEX worked very well to shift the protein. If that does not work perhaps you can look at your expression conditions. If the protein has a problem folding then a lower expression Temp. and/or longer expression time may work better.

Best of luck,

Chris.

Hi Michael,

I had similar experience while co-expressing Mycobacterium protein with GroEL/GroES chaperone system. I could separate them to some extend using IEX and ammonium sulfate precipitation. Of course, I could not separate them completely and I had a huge loss of POI.All the above mentioned methods would help to some extend to reduce the amount of contaminating chaperone. But in my experience, whether it be chaperones, cleaved- GST tags or cleaved-MBP tags, once they form tight complex with your protein, it is hard to rescue your protein completely out of their claws. The final purpose of purification should guide for the selection of solubility tags / chaperones !!!!..

Good luck with your separation. Hope you will share your success story.

SAI

Hi,

I agree with all the above approaches, but wanted to add that we once got lucky by just lowering the salt concentration in our purification buffers. Anything you can do to keep the POI folded will help to dissociate the chaperones. ATP addition also worked for us.

David

Incubation of the protein solution upto 5mM ATP and1mM Mg O/N followed by affinity chromatography should work. also do add ATP and Mg in all your buffers.

Please describe the purpose of your purification (assay or antigen generation or…). There are nice (usually harsh) methods to avoid protein- protein interactions. Please describe the tag and the buffers you are using, hope we will find the solution;)

We would like it for multiple purposes: antigen for antibody generation, substrate in a kinase reaction, in vitro binding reaction with potential partner proteins). The tag is 6xHis, non-cleavable. It is purified in 50mM Tris pH 8.5/200mM NaCl, 1mM b-mercaptoethanol, 100mM imidazole.

Sorry to say it, but you did a good job. Just this complex is formed in bacteria, to be honest, when 200mM salt is not affecting it, I see a long way before you. What I would do is to set-up the salt concentration could destroy it. I equilibrate Superdex 200 size exclusion column with the buffer with high salt (0.5 – 2M) and run my sample in original buffer. The desired shift will tell you the way you should follow (try also ATP or Mg as others suggested). You don’t have detergent in your buffer, but I don’t believe this would help for the strong interaction you have. For antigen the solution is very simple – do the purification under denaturing conditions slice it out of the gel, it will be ready fast.

We have developed a method to remove HSP70. In our group we have also successfully applied this method to remove chaperonins. If your protein has not reached a sufficient degree of folding to stay in solution, probably chaperon removal will induce protein precipitation. Since the method is simple, it can be tested. The protein solution of interest is incubated with E. coli proteins previously heat treated in the presence of ATP, so that an exchange takes place between the protein of interest and the other proteins. You should have your protein with some tag (i.e. His-tag) to recover it. The treatment can be also be done on the affinity column before elution of your protein. The reference of this work is Rial and Ceccarelli Protein Expression and Purification (2002) 25, 503–507. I can send you a copy if you wish.

Thanks Eduardo! Just downloaded a copy and will read it immediately. Great advice!

Just got the mass spec results back. The protein is NOT a chaperonin but it is ArnA. I know there is a modified BL21(DE3) strain where ArnA is fused to chitin binding domain and can be removed via chitin chromatography. but we used arctic express cells for the chaperonins that they have so we cannot easily change. Has anyone had experience removing the ArnA contaminant by ion exchange chromatography? The pI of ArnA is 6.39 and our protein's pI is 4.81 so it seems like ion exchange should work.

Michael, It is an interesting coincidence! We have also the same problem a couple of years ago purifying chloroplast Hsp100 chaperones expressed in E. coli. ArnA is repressed by bivalent cations. So, try to express your protein using the E. coli BL21(DE3)pLysS (or any other similar strain) adding 10 mM MgCl2 to the culture medium during culture and protein expression. Adding MgCl2 solved the problems for us. Good luck!

Brilliant!! Will give that a try.

I don't work with ArnA, but I had cases , where  I had 1:1 chaperon to expressed protein. After affinity chromatography I used Q sepharose HP and I got rid of chaperone, because it binds tighter to the Q, than my protein of interest.

It elutes between 21-24 mSiemens.

Good Luck

Hi all,

For Eduardo A. Ceccarelli: at which temperature do you wash the resin with the denaturated protein/ATPMg solution? RT or 4C (cold room)? Thank you.

Best,

N

If the protein resists 25 C, I would wash the resin at that temperature. I suppose it is necessary that the HSP70 has some activity to be able to interchange substrates. Low temperature may decrease the activity of the chaperone

Regards,

Eduardo

It makes sense! Thank you very much for your quick response.

Best.

N

Hi all! I have a question about preparing the denaturated E. coli proteins. How exactly the proteins are prepared? Is this just crude extract of E. coli cells without expression vector?

Hi all,

For Eduardo A. Ceccarelli: Great answers! But why addition of Mg2+ can repress the expression of ArnA? Any other host cell protein can be repressed by divalent cations?

Best regards,

Hao

I guest that it will only work on bacteria containing the PhoP-PhoQ two-component system. This system is found in intracellular pathogens, and we are lucky that it is also is present in E. coli.

Regards,

Eduardo

Concerning the Barbara Ramsak question: "Denatured E. coli proteins were obtained by heating an E. coli lysate (2 mg/mL protein

concentration) in 50mM Tris–HCl, pH 8.0, 150 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride for 10min at 65 oC, centrifuged

for 10 min at 12,000g, and the supernatant (0.15 mg/mL protein concentration) stored frozen in aliquots for future use".

Thanks, Eduardo

hello all,

i find this discussion valueable, because i'm in same kind of trouble these days.

i expressed mycobacterial protein in E.coli arctic axpression cell line.

Protein of interest is fused with MBP and His-tag on N-terminus(total 85kDa size).

PI of protein with tag 6.70

buffer tris ph 8.0 50mM, Nacl300mM, glycerol 10%, pmsf&Bhcl both 1mM, BME 3mM.

i tried to purify protein of interest from Ni-Nta as well as MBP binding resin.

but i failed in both tries beacause i am getting my protein in flow through and mainly 2 bands in elution sized around 62 kDa and 50-55 kDa.

i want to share sds Gel pictures

gel pic 1st ni-nta affinity chromatography- lane1marker

lane 2 flow through

lane 3-7 wash

lane 8-13 elution

amylose resin chromatography

lane 1 marker

lane 2 flow through

lane 3 wash 0.5mM maltose

lane 4-9 elution 5mM maltose.

i got expression in plys cell line as well, should i try this to get rid of these impurities?

How to add Mg ATP? in which concentration? are their any specifications in this regard