Hello Basavraj,

From your description of the problem I would suggest to play around salt (increasing) concentration in your buffer. Otherwise without knowing any specifics of the protein, it is difficult to suggest any further. You might have to think of designing a different construct of this protein etc. Did some bioinformatics analysis, if it was not done so far, you might get some useful information. Good luck.

Andrei

My protein is a secretory toxin protein in nature......Initially I have tried play with salts in dialysis buffer...But all time I used to get some or most of aggregated proteins in DLS results without any specific  trends.....This protein is highly temperature sensitive... Yup... I have tried some bioinformatic analysis....This protein seems to be having some disorder regions...Some localized charged amino acids....N terminal half of protein is having pI around 9 and C terminal of protein having pI around 5 and overall pI of protein is neutral.... This much only I Know about my protein.. Probably I need to do more bioinformatic analysis...Can you suggest me... some more tricks for understanding. Thanks in Advance..

Have you thought about expressing N- and C-terminal part of your protein separately?

Alternatively, you could try nanoDSF. Due to it's ultra-high resolution, you can easily detect multiple unfolding events, cause by unfolding of individual domains of a protein. You might also be able to see dissociation of the higher oligomers into monomers. With nanoDSF you can screen 48 conditions in parallel for the best conditions where no oligomers are present.

Please find some further information following the link below.

http://www.nanotemper-technologies.com/technologies/nanodsf/

I see, secretory toxin, it sounds that you might need to think of figuring out a potential interaction partner for the toxin (if any) for co-expression, i.e. either a small molecule or a protein (host or native, i.e. from a bacterium). Otherwise try co-expressing the toxin with GroEL-ES, for example. I am not sure where and how big these disordered regions are but they may causing problems. If some of them N- or C-terminal remove them by mutagenesis, of course think of the toxin function first, i.e. if this removal would not cause any additional problems. Heide did suggested an approach in getting two parts of the toxin separately cloned and purify. How big is the toxin?

Protein is around 43 kDa....Yup it do have N terminal disorder region....I am thinking exclude it...as it does not have any function...Its binding partners are not know till now...I have solved intial expression problem by shifting to very low temperature and low IPTG for induction....and I have managed to get monomer of protein too....My next step is crystallize this protein....I don't feel confident to follow crystallization trials of this protein as we do for normal stable proteins...I know that it can't tolerate small change temperature  nor rigorous change in buffer conditions...such as salts or additives etc...

the N-terminal region may still be important - be careful with what you exclude. I would guess it is important for secretion, i.e. interacting with the secretory machinery or perhaps some interaction partner, a chaperone within the bacterium's cytosol. Look for neighboring genes of this toxin - it may help you to find any clues. Also, if you know the function of this toxin try co-crystallization with some ligands etc.

I have tried with its ligands.....it still forms higher order oligomers........Probably I need try once more...Thanks for your advice....

It's not uncommon that proteins crystallize at rather low concentration. What is your starting concentration in crystallization trials? You may try lowering it.

Also, try to use some additives, like mild detergents, urea etc. You may first try size-exclusion chromatography/DLS in their presence before starting crystallization trials.

You may try partial proteolysis of your protein to detect any stable domains within full-length protein. Sometimes, it works just to add some proteases directly in crystallization conditions (for example trypsin at 1:1000 ratio).

Another suggestion is to try reductive methylation in case your protein tolerates the procedure. http://www.nature.com/nmeth/journal/v5/n10/full/nmeth1008-853.html

To Anatoly Dubnovitsky...

I use 6-9 mg/ml protein concentration for crystallization trials....Even at low concentration protein do forms oligomers ( almost 80-30 % depending on buffer  composition)... probably oligomerization either happens during purification or with in cell....Does using detergents won't affect its function or structure..??.Yup i'm thinking about partial proteolysis too...Thanks for your interesting suggestions...

Protein concentration in crystallization trials can be down to 1-2 mg/mL and sometimes even lower.

Low concentrations of detergents normally do not affect the structure nor function. We are talking about octyl-glucoside for instance, dodecylmaltoside etc., not SDS-type of detergent. The membrane proteins are routinely crystallized in the presence of different detergents. Non-membrane proteins are often purified in the presence of mild detergents to make it easier. As a reference point you can check formulations of commercial Additive screens for crystallization.

The presence of two distinctly charged parts may promote oligomerization. In this respect reductive methylation may actually change the behavior of the protein. Theoretically...

@Anatoly Dubnovitsky....Yup methylation is good suggestion..buttechnique for our lab...probably i need some time to try and standardize it......

As you said two distinctly charged parts may be likely cause oligomerization....then probably by using solute or solvent with particular charged nature only ( either positive or negative )oligomerization can be prevented theoretically, as it will only binding to one site of protein and hinder oligomerization ....Does it will work????? Can you suggest  me some such solutes? or solvents?? I am not getting examples of such solutes or chemicals....

Toxins tend to form oligomers specially because they have an interacting partner like anti-toxin. Is there something like that known for your protein? Alternatively you can try playing with NaCl concentration and arginine in buffer. You can also use urea or Guanidine HCL in low concentrations. 

@Sudhir Kumar.....Yup i have tried playing with NaCl....With increase in NaCl concentration hydrodynamic radius of protein tends to increase....I believe my protein is likely to have some binding partners inside the host but not yet identified....Urea or GdHCL is some thing i need to try....But I have some doubts....Adding this denaturant may create some conformational heterogeneity in proteins.... this may further hinder protein crystallization process...Thanks for your useful comments...

Hi Basavraj

I would try the opposite : increase the concentration to select and stabilize the oligomer and try to fix it with a cross linker (ie glutaraldehide...). And if it doesn't work try SAXS http://www.ncbi.nlm.nih.gov/pubmed/25404693.

good luck

Hi Basavraj,

My proteins also forms higher order oligomers and DLS reading suggests that its a homogeneous aggregation. Could you please tell me that how did you manage to get the monomeric form of your protein. I add Arg and MgCl2 in my lysis and GEC buffers. However, i have not been able to suppress aggregation.

Please let me know if you have used some other additives to get the monomers of your protein.

Thanks & Regards

Sunil Singh