hydrophobic interactions are the most prominent factor in folding of proteins and help them attain their energy minima during folding. however, whenevr a "violent" method like sonication is used with cells or cell extracts, the energy minimum of a protein is overcome and it unfolds, sometimes exposing a significat proportion of its hydrophobic regions, and leading to aggregation. treatments like ammonium sulfate bring about the same unfolding but prevent aggregation due to their kosmotropic nature. Inclusion of a small amount of a kosmotropic salt during release of proteins from membrane compartments definitely helps. if you dont want salt in your extract you can try something like glycine. inclusion bodies as suggested by others here are definitely a problem, but i have consistently found that if you can control aggregation during homogenization, most of your worries are done away with. you can alternatively use a bead based homogenizer instead of a sonicator. as for the first thing you asked, yes, if proteins are aggregating, they would definitely be found in the pellet instead of the supernatant. i would like refer you to a research paper that deals with these issues quite extensively:

Bondos and Blicknell (2003): detection and prevention of protein aggregation before, during and after protein purification. Analytical Biochemistry, 316: 223-231.

It sounds like your protein is either insoluble or it is binding to insoluble factors in your prep.

Dear Santosh,

There is no chance for the pelleting of soluble protein during centrifugation. Because soluble form of protein will not settle down.

But due to heat generation during probe sonication will denaturate protein and it get converted to insoluble nanoparticles that will settle down during centrifugation.

What is your purpose behind sonication?

Its bath sonication or probe sonication?

Hi Santhosh,

Your protein of interest is insoluble, either it is in IB's or its a membrane protein. play with different types of detergents in lysis buffer. did you resolve the first supernatant obtained after first spin?

Regards,

Poornima

Thanks for your explanations.

I'm doing sonication by using probe at 35% amplitude(30 sec on/off).

What I have to do if it is binding to insoluble factors in my preparation?

I have not resolved my first supernatant,basically its a cytosolic protein......

Any soluble protein has limits of solubility, i.e. the maximal concentration at which protein is still soluble. When you spin your sample all the protein in the solution concentrates at the bottom of the tube due to the protein's high molecular weight. This, in turn, causes a rapid increase of the LOCAL concentration of the protein, which may lead to its precipitation if that LOCAL concentration is higher than your protein's solubility limit.

Try using higher volumes of buffer to solubilize your protein from cell lysate. 5-10ml of buffer per 1 gram of pellet should be enough.

My protein have mol.wt of 36kd,can I proceed for purification with the first supernatant...?

What you can do is a high-detergent wash such as Sarkosyl - try these refs. Sarkosyl works very well in my experience:

http://www.biomedcentral.com/1472-6750/12/95

http://www.biotechniques.com/BiotechniquesJournal/2010/January/Purifying-natively-folded-proteins-from-inclusion-bodies-using-sarkosyl-Triton-X-100-and-CHAPS/biotechniques-185876.html

Thanks Mr.Emil Bulatov...

I have used 15ml of lysis buffer per 1gm of pellet.

Mr.Gary McDowell

I have tried with 3% sarkosyl but still it is not solubilizing........

Try immediate buffer change after the first centrifugation on the size-exclusion chromatography column. Choose a gel in a way so that you have your protein come out in a "free volume", leaving all the proteases and degradated protein fractions "behind".

For my understandig it is completly impossible to pellet soluble protein, the only thing you will get is a gradient inside your tube, if you centrifuge over a long time. You have only written that you have used 15000rpm, more importent to know is the force in xg you have used. Did you see any turbidity before centrifugation, if you are not sure you can measure in a photometer @ 600nm against your buffer as reference.

Is it natural protein or is it a recombinant expression of the protein as a fusionprotein? Normaly the tag of a fusionprotein should prevent precipitation.

If a precipitation happens, this depends on different parameters like pH, salt or temperature. Some of this parameters you can exclude during centrifugation, but if the temperature change during centrifugation, it could be that your protein precipitats. If you try to resuspend your protein the next steps depends completly on what you will do with the protein next. You can use chaotrops like urea, thiourea or guanidiniumHCl, or you can use different amounts of salts, or differnt detergents especially SDS (which should work, because otherwise your SDS-PAGE show no result) to resuspend the pellet.

-It is a recombinant protein contains his-tag at its N-terminal........

It's only a his-tag? directly fused to the protein sequence? whithout any other aminoacids? So if you will use Ni-column you can use different detergents and chaotrops. You should take a look in the manual up to which concentration you can use this chemicals and try to resuspend the pellets.

Presence of the His-tag makes it even easier. Then you can perform a buffer change on the Ni-column. Just load your sample after the first centrifugation, wash with a good amount of the buffer and then elute using Imidazol.

I will try it Mr. Artur..........

Its is fused protein with his-tag and some amino acids are there in between tag and my protein of interest.

Sometimes having the protein in the pellet is good because it is then quite pure. Wash the pellet with native buffer. Then dissolve the pellet in 8 M urea (if possible add 1 mM EDTAand 5 mM B-Me). refold the protein by diluting (~50X) in a buffer. This should work. if you need higher conc of protein then you can concentrate it by using 10KD Viviaspin filters.

For a detailed protocol about how to purify protein from pellet you may look at this paper.

http://www.ncbi.nlm.nih.gov/pubmed/19233290

Thank you, kanchan........

Is it possible that the cells are not crushed? If that's true, you will see proteins in the pellets and not in supernatant.

What kind of buffer do you use for resuspension of the bacterial pellet before sonification? For my understanding a concentration of 1gram of bacterial pellet per 15ml resuspension buffer could be to high, if you have a very high induction and thereby a high concentration of your protein of interest. You can try to use more resuspension buffer. If you use standard e.coli for expression, my experience is to sonificate on ice 10times 12sec with a break of 30sec in between to cool down the suspension.

Lysis buffer: 50mM Tris(pH 7.40),150mM NaCl,Triton X-100 1%,Lysozme and Dnase (50 mg/ml stock each),1mM PMSF, 5mM DTT,2% Glycerol.

Your protein is expressed and precipitates when you spin down cell debris. That means that either it goes to inclusion bodies during expression or you lysis buffer makes it insoluble. Check the pI of protein and try to charge it playing with pH of your buffer. If this will not help use weak detergent to solubilize the protein. You may also try to resuspend cells in the lysis buffer and flash freeze it in LN2. Then use gentle technique to break up the cells. How long are you sonicating your cells. Common mistake is to sonicate your cells too long and overheating your sample. By centrifuging you cannot spin down soluble proteins. Unless you chemically precipitate them

Thank you..Michal Boniecki

There are 2 major techniques to get higher ratio of soluble protein compare to IBs, and it is lower concentration of inducer and lower temperature.

Iam inducing my protein at 16c and 0.4mM IPTG.

You did not mention what host you are using, if it is e.coli then most likely it is not IBs but I would try lower IPTG, around 0.1mM.

If it is not IBs then most likely the cell rupture was not effective or your protein bound to membrane fraction.

You can try precipitating the soluble protein using ammonium sulphate gradient in your sample before centrifugation.

I have expressed in E.coli BL21 DE3 strain..........

In addition to Michal Boniecki's recommendations to avoid protein precipitation, consider including cofactors if your protein requires any. I spent some time purifying glycolysis enzymes and 4 of the 12 required cofactors to remain soluble during purification. For example fructose bisphosphate aldolase required Zn2+ at 0.1 mM.

Another possibility to consider is that your protein of interest associates with insoluble components of cell debris.

hydrophobic interactions are the most prominent factor in folding of proteins and help them attain their energy minima during folding. however, whenevr a "violent" method like sonication is used with cells or cell extracts, the energy minimum of a protein is overcome and it unfolds, sometimes exposing a significat proportion of its hydrophobic regions, and leading to aggregation. treatments like ammonium sulfate bring about the same unfolding but prevent aggregation due to their kosmotropic nature. Inclusion of a small amount of a kosmotropic salt during release of proteins from membrane compartments definitely helps. if you dont want salt in your extract you can try something like glycine. inclusion bodies as suggested by others here are definitely a problem, but i have consistently found that if you can control aggregation during homogenization, most of your worries are done away with. you can alternatively use a bead based homogenizer instead of a sonicator. as for the first thing you asked, yes, if proteins are aggregating, they would definitely be found in the pellet instead of the supernatant. i would like refer you to a research paper that deals with these issues quite extensively:

Bondos and Blicknell (2003): detection and prevention of protein aggregation before, during and after protein purification. Analytical Biochemistry, 316: 223-231.

Thanks Mr.Samir Sharma

All the above suggestions could potentially help to counter your problem, once you identify it!

If soluble protein levels are reasonable at the time of expression, your lysis method and lysis buffer could have an effect on aggregate formation post-lysis. Playing with the buffer components could help. The key factors are:

1) pH (atleast 2 units lower/higher than the pI of your protein),

2) buffer (Tris, HEPES, bis-tris etc. depending on the pH you want to achieve),

3) salt concentration (higher concs. lower hydrobhobic interactions)

4) Additives (optional): these could be detergents like Triton, or even the amino acids, such as glycine and arginine which are known to lower protein aggregation. (bear in mind though that the presence of arginine and glycine in your lysate supernatant can significantly hamper binding to Ni-NTA resin).

BUT there are other things you could do at the construct design and cloning stage of your work and is actually less time consuming than optimising expression and purification conditions. I don't know whether you're after the full protein or just a domain, but using a multi-construct approach can help you find one that expresses much better than the others! Even with the same protein, different constructs with different amino acid sequences at either end of the polypeptide chain can affect their properties. you need to pay attention to your protein sequence during construct design.

(doi: 10.1016/j.pep.2007.11.008.)

Try cloning the sequence of single domains, or several truncated versions of your protein. Perform a small scale expression (I do 50mL) at a moderate IPTG conc (0.1-0.5) and a low temp overnight. lyse a small portion in a microcentrifuge tube and analyse the supernatant and pellet by SDS-PAGE to get a general idea of expression levels. Scale up the ones that seem okay straight away and purify one by one. I followed this approach with my work, using the same expression and purification protocols and with my 7th construct I got a protein fragment that's worth taking forward to purification optimisation. good soluble expression levels make your life easier!

Another less time consuming alternative than the ones above (and you could try this first) is to simply clone your construct into a plasmid with a cleavable fusion tag, such as MBP. It could enhance the solubility, yield and also lower the toxicity of your protein as it assists its folding during expression. There are papers on this, and it worked in my case for a human protein that did not even have high expression levels as IB!

(Protein Sci. 1999 Aug;8(8):1668-74.

Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused.)

I hope this could help you! good luck :)