Adding to Helge's response, if this protein does require high ionic strength to stay soluble, consider using an arginine-glutamate buffer. It has low conductivity which will maintain good signal-to-noise in an NMR experiment. In my experiences, 50 mM L-arginine and 50 mM L-glutamate can drastically improve protein solubility.

http://www.sciencedirect.com/science/article/pii/S1090780707003783

A StrepII tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys), this is provides a small tag (N or C)which can be purified under very gentle conditions and the resin seems very inert with little other contaminating protein binding to the resin, we currently use the IBA high capacity resin but there are other suppliers

Just my 2 cents worth suggestion. You may want to try the new Fh8 tag system. Sounds interesting. I have attached paper for your reference

If your Protein is unstable or "semi-stable" you might also try to optimize your buffer conditions. pH and salt concentration can have a high impact on Protein solubility (particularly because you have a highly charged Protein) Of course this is a cumbersome process but worth it if you have problems with unspecific protein aggregation in  ITC and NMR experiments.

Adding to Helge's response, if this protein does require high ionic strength to stay soluble, consider using an arginine-glutamate buffer. It has low conductivity which will maintain good signal-to-noise in an NMR experiment. In my experiences, 50 mM L-arginine and 50 mM L-glutamate can drastically improve protein solubility.

http://www.sciencedirect.com/science/article/pii/S1090780707003783

Have you checked the isoelectric point of your protein? If the protein has a PI of 8 and your buffer is pH 8 then the protein won't be charged properly and therefore crash out of solution quite quickly. Always choose a buffer system that is about 2 points away from the PI of the protein (e.g. for PI = 8, choose MES, BIS-TRIS or BIS-TRIS propane, all of which will buffer around pH = 6).

If an N-terminal His-tag doesn't work, maybe the problem lies with the position of your protein's N-terminus, which might be somehow disrupted by the tag or the tag interacts/occludes the active site. In these cases changing to a C-terminal His-tag often works.

As Helge and Justin already mentioned, highly charged proteins usually need buffers with high ionic strength. For structural biology we tend to keep our proteins in buffers that have very low NaCl concentrations as NaCl (which is a chaotroph) can inhibit crystallisation, but if the protein needs 200 or 300 mM NaCl to stay in solution, then you should at least try and see if your experiments still work with such a buffer. If this is not an option you should investigate other buffer systems like the argenine/glutamate mix mentioned by Justin.

Could you be more precise concerning your use of the word "unstable" ?

Do you mean that it is very quickly degraded, or is you protein becoming insoluble after Tag removal ?

If it is an insolubility problem, Antonio's comment will be very useful to you...

Hi all

thank you so much for all your suggestions. I will consider all of these and try to get a stable protein. C terminal his tag was tried too but in case of my protein, if I add a tag at the c terminus, it becomes unstructured. So currently I'm trying n terminal tags.

My protein is soluble but it's quickly degraded once purified. 

I will try all your suggestions to check what works out for my protein

200-300 mM NaCl will cause sever problems for a cryoprobe. I'd try these in this order

1. f the pH is near the pI this is an easy fix.

2. The arginine/glutamate buffer is a very interesting idea as arginine is strong promoter of protein folding. The concern would be the contamination of the signal from the buffer. The publication says this is not  a problem for 3D NMR, however it may make optimizing by 1H spectra difficult.

3. 20% glycerol will also help fold many proteins. The disadvantage is the cost of deuterated glycerol and the increase in line broadening due to the increase in viscosity.

Thank you very much. I will try these suggestions and let you know of my progress

Another question about the meaning of 'unstable'. If it's insoluble, then a number of fixes have been suggested. However, if it's unstructured you should consider the possibility that this protein is intrinsically disordered. Highly charged proteins often are. If this is the case, you can try to force folding but the structure you will get will not be a native one.

The protein is soluble but not stable. I speculate that it might not fold properly. I will try to find out more in this regard.

Thanks

how you define the stability of your protein ?

Does it gets degraded  

How did you get on with this  Prabhadevi?

I had issues with GST tagged protein precipitating when I did the on column cleavage.  In the end I had to use 500mM salt to keep in solution which gave pretty poor S/N (600mhz cryoprobe).  I switched to the magic buffer that Justin has suggested above which gave much better S/N and kept the protein in soln.  The glu/arg does show up in 1D spectra but for  2D/3D 15N HSQC based experiments it was fine.