Hi,

Have you tried adding reducing agent?

I think the salt is causing precipitation of the protein (salting out effect), so you can try lowering the concentration of the salt.

You could also try changing the pH.

@Vu to: I am using β-ME in the wash buffers.

@Khaleel: I have lowered the salt concn. upto 150mM. but at lower conc. the precipitation is much faster.

@Sebastian: sir, the protein is not well expressed at lower temp. (16 and 25 deg C). In addition, the precipitation is so rapid for the initial elutions that I am not able to process it any further.

@Adam: sir, I have tried pH: 5.6 and lower but I havn't used a pH higher than the pI that is because silica beads (Ni-NTA) are not inert beyond pH 8.4.

If you want to try an alkaline pH for string the protein, when you dilute the protein after purification, use a high-pH buffer that will bring the pH up to the desired level.

Dear Suraj

You can try purification at room temperature some time at lower temperature thermophilic protein get precipitated.

Apart from that you can lower the salt concentrations.  

@bibhuti: I already have tried purifying the protein at lower temp. and with lower salt conc. but it precipitated faster compared to purificaton at 4 deg and with higher salt conc.

@Adam: Does the interaction between two different pH buffers will hamper the protein stability? Is it feasible to add two different buffers (For e.g. Pipes 6.6 with Tris 8.8) ? I guess this may show some adverse effect while crystallization or during biophysical characterization.

Dear Suraj

I suggested you to use higher temperature like 25 or 30 not lower than 4.

That may work for thermophilic protein.

I have never heard of a problem with protein stability being caused by using a mixture of 2 different buffers. For crystallization, it would probably be better, for the sake of reproducibility, to dialyze the sample into one well-defined buffer once you have identified conditions in which it is stable.

Thanks everyone for responding.

@Adam: Sir the idea of diluting the protein with a higher pH buffer worked but when I treated the protein with EDTA (2/5/10 mM) for demetallation, the precipitation was observed again.

Would be thankful if anyone can help me out with this.

I don't understand when you say "@Adam: sir, I have tried pH: 5.6 and lower but I havn't used a pH higher than the pI that is because silica beads (Ni-NTA) are not inert beyond pH 8.4."

Why not try something more neutral 7 or 7.5?

I wouldn't lower the salt concentration during purification, but would dialyze right after the Ni-NTA since Imidazole is known for destabilizing protein structure.

Suraj, When you added EDTA to the protein that was diluted in higher pH buffer, did you use EDTA that was at the same pH as that buffer? EDTA is acidic, unless neutralized with base.

Hi Suraj

Try to purify the protein at 20 or above temperatures(as it is a thermophilic protein).

All the best

@jayashree: Ma'am, I have tried purifying the protein at 25° C but it didn't work. It is recommended to heat the lysate at 70°C for thermophilic proteins to remove the E. coli proteins but in my case, the protein of interest also got denatured while heating.

@Adam: Sir, I can try changing the pH of EDTA similar to the protein buffer. Any recommendation for the concentration of EDTA to be used? Usually, 5-10 mM is recommended.

Suraj,

Is the target enzyme dependent on a metal (e.g., iron) or any other divalent cations, etc.?

You could also try different salts, reducing agents, and so forth.

Also, how pure is it off of the Ni-NTA? Are there still a decent amount of contaminant proteins that could be contributing to your problem?

EDTA is used to chelate metal cations, which helps prevent proteolysis by metal-dependent proteases, heks prevent metal-catalyzed protein oxidation, and binds up any nickel that came off the Ni-NTA column. I would use 1 mM.

@Adam : So as per your suggestion 1mM EDTA can be used in N-NTA column without stripping the Ni2+ ions? 

Even after purification, proteins are susceptible to proteolytic degradation? This means tputting 1mM PMSF and EDTA both in all the buffers could solve the problem?

@kanti: EDTA is a very strong chelating agent. Adam didn't suggest to use EDTA in Ni-NTA column. It is to be used post purification. What he suggested is to use 1 mM EDTA at the time of demetallation.

@suraj. Thanks suraj. 

actually I am also encountering the same problem of protein aggregation after purification. However my protein is a small globular protein of 20kDa. I have tried with 50mM Sodium phospahte, 300mM Nacl, reducing agent, glycerol, chaotropes etc but nothing stops it from aggregating. Do you have any suggestions. 

If now the problem is EDTA, try not to use it unless the presence of metals is a problem for the next experiments. PMSF can inhibit proteases.

Furthermore, I used Ni columns at pH 8.5 without problems. Yo can try to use this PH along the purification.

Imidazole is known to destabilize the proteins in some cases. I would try to elute the protein from Ni-NTA with EDTA or with low pH (4 or 5, if the protein is table at low pH), in this way the protein would not be in contact with imidazole. I observed in some cases that this improved the protein stability after purification.

Hi Suraj!

What protein are we talking about? If your protein is a thermophilic one, you can try to purify it by high temperature, we say 70 °C or more, the rest of the protein will precipitate and your protein may not! In that case take care of the temperature dependence of the buffer you use,  I recommend a simple phosphate buffer. In any case pay attention of the pH! because your protein has a pI near 7. Other thing you could try is purify the protein in denaturing conditions and then refold it. Good luck!

@Diego: Thanks for responding. I have already tried purifying the protein in 70 °C but I didn't get any protein. Although I haven't tried using a Phosphate buffer. I have purified the protein successfully in Pipes buffer pH 6.7 without precipitation. The protein is getting precipitated after adding 10mM of EDTA (mentioned in the above comments). Any particular pH you would like to suggest for a protein having pI 7.7 to be purified using Ni-NTA chromatography?

Suraj, 

It is not rare that the thermophilic proteins make use of metal ions to be stable, maybe if the precipitate after the addition of EDTA is because you are removing the ions. Why you add EDTA in any case? The pI of the is a real challenge, an option to avoid the use of Ni-NTA chromatography is purifying by  IEC using  a captoQ or captoS exchange chromatography and stay away  the pI