Dear Rajivghandi,

I think the best option would be to change the Tag to His6, Halo or MBP, but I guess you already tried this and it's not suitable for your protein. Maybe it already helps to change the protease cleavage site between GST and your protein of interest, so that you obtain a more specific elution of your protein from the on-column cleavage.

Do you really have to express as a fusion? All too often fusion and tag systems are used for overexpression unnecessarily. In my opinion, this usually only complicates collecting high-quality material for structural studies.

Hi there,

Pass your eluate containing the mix of proteins onto Glutathion-agarose column again to trap the free GST. Hopefully your protein of interest will be in the flow through.

Hi Rajiv,

The good news is "your protein is good for crystallization". It just needs a few tweaks during purification to get rid of traces of GST. Do not worry about the theoretical PI, it is always a distraction. No protein is 100% pure, it just depends on how much you load and the sensitivity of detection. Now for the clean up:

1. Extensively wash the column before cleavage to get rid loosely bound fusion protein.

2. Hold the NaCl gradient around your fraction 3 (mono q column) and this should help remove more of GST. You will lose some of your protein, but that is the compromise.

3. Use a S75 as your last step. Your protein is likely a dimer as it is eluting with GST(GST is a dimer). The S75 will give you a slightly better separation in the size range (15-40kDa) but not by much. Do not expect baseline separation (your proteins are almost the same size).You may not need this if your mono q works slightly better.  

Hope this helps,

Good luck with crystallization(this is the tricky part now).

Dear Katrin Bagola thank you for your suggestion

i have tried on column cleavage but in flow-through i got both the protein coming together so i again passes through fresh GST but still the faint GST is still there. 

Dear Dominique Liger

thank you for your attention 

i have tried this method already, please see the image i have given here with this i have set up for crystallization i have got crystals in many conditions but morphology wise not looking good 

Dear sujay  thank you so much.

i have already try to purify this with S75 but in that co-elution was happening.

 i pooled the those fraction having less GST lower band and i concentrated to 15 mg/ml and i set up crystal screening with 8 different kits.  i got crystals in more than 60 conditions but morphological not looking good (leaves like structures). so i thought that may be increasing purity i could get 3D crystals. so i m planing to change tags. please look at the picture i provided. 

Dear Rajivghandi,

Are you sure that the lower band is GST contamination? I mean did you do mass spec? Actually for me for me it seems rather a degradation..if you carefully check the first gel which you uploaded the contaminant seems to migrate higher when compared to GST (see fraction 5). Also notice that in lane 6 you actually have two bands (contaminants). I would suggest to destain better your gels in order to see clearer what you have there. On the other hand in the second gel you have a number of contaminants which also indicates that your protein is prone to degradation.

Regarding the purification on MonoQ, it is hard to believe that you can't separate your protein from GST. MonoQ column is very high resolution column therefore if you do a proper gradient, for sure you will be able to separate it. As a control you can add back GST to your protein and then load on MonoQ and perform a proper gradient in order to get two defined peaks and then check your protein. If you still have this lower band that's a good indication that is a degradation.

The S200 column was not a really good idea as it doesn't have the necessary resolution required in your case, not even the S75 in my opinion, therefore the only option is to play with the MonoQ (i.e the gradient).

Before going for recloning I would suggest to do limited proteolysis, perhaps this way you will manage to identify a more stable fragment resulting better crystals.

Good luck!  

Agree with most of the comments above regarding purification (notably the last ones from Csaba).

Would just have a general comment: you told you were having already 60 conditions giving crystals ! So you just already made the job from crystal screens and your protein looks to be suitable to crystallisation (even if not perfect) !

What you need to do now is to check the conditions corresponding to these 60 hits, and optimise your crystallisation condition for your favourite protein !

Also, remember that the way crystals look like doen't tell you haw they will diffract ! I saw some tiny crystals looking bad that were diffracting to 2 angtröms, and some big beautiful ones diffracting very poorly... Only the diffraction can really tell you, so test it as soon as possible (will at least tell you if this is salt or protein, which is a good point of start).

Good luck

Thank you dear Csaba and yann your comment is really usefull for me  i wil try 

if you reduce the NaCL down to 150 by dialysis or de-salt and then run  a S200 SEC the GST should form a higher MW oligomer and you should get reasonable seperation

Dear james thanks for your kind attention and answer.  the buffer i used for S200  had 150 mM NaCl, Tris Hcl.pH 7.5 , EDTA 1mM DTT. But in SEC i expected that GST form dimer anf my protein of interest form trimer but there a coelution.of GST dimer with my protein trimer...