Dear Jorg

thank you very much

It is a soluble protease protein from the plant source. In plant cells this protein moves in and out and according to the literatures, this is the soluble protein. As you said I will analyze the amino acid sequence.

while doing Expression and solubility test, please do the cell lysis using Sonication. That will give a clearer result. Also still, if it seems in the cell pellet that optimize the growth condition using traditional methods like temperature of induction, inducer concentration etc. Also try different expression strains. There is one strain called SOLUBL21. It claims solublize the protein. Also you can try to co express with different kind of chaperones. You can also try to add 100 mM L -arginine or glutamate during cell lysis. If it still comes into pellet then its not a surprise as most of the research in soluble protein is hampered by this problem.

Don't overlook trying different lysis buffers, various pHs, and different salt concentrations. It may be that your protein is just not happy in phosphate buffer. Maybe try 100 mM Tris or HEPES with 100-400 mM NaCl. As a last ditch effort you could add 10-20% sucrose to your lysis buffer. Sucrose is commonly found in protein therapeutics as a stabilizer.

thank you Bradley and Ranjan. I will really try your suggestions....

10mM Tris-HCl buffer pH must be checked with the protein's pI plus add 500mM NaCl 5mM BME. But it depends on the protein. Since I do to know what the protein is, could not suggest/add besides already discussed here.

Hi Sharmila,

There are many things you can do to try and improve its solubility. Assuming it wants to be soluble (ie you dont need a new construct to make it soluble) then modifying the lysis technique can make a big difference. Lysis by Lysozyme is one of my preferred ways to gently extract a large protein (I regularly work with 60-120kDa proteins in bacterial cells). If the protein can handle harsher methods such as sonication or cell disruption than this is probably a better lysis technique.

As Andrei and Bradley suggested changing your lysis buffer can also yield a good improvement. Whilst the buffering agent (Tris/Phosphate) is important it is often the pH that is more important as well as the additives such as 150-500mM NaCl, 5-10% Glycerol, 0.5-5% TX100 that help a protein remain soluble and stable in solution.

Good luck!

Russell

Hi,

I agree that optimizing your lysis buffer can improve yield dramatically. Besides changing pH and NaCl concentration (I typically test 0, 100 mM, 250 mM, 500 mM and 1M), also the type of detergent can be essential (try 1% NP-40, or 0.5% CHAPS). If that doesn't help, you can try making a different fusion (using a His6-SUMO tag often solubilizes proteins, or MBP).

Good luck!

Goedele

1. Do you know for sure that cell lysis is complete? I assume you are using bacterial cells. You may have a large proportion of cells still not lysed. This, surprisingly, can happen easily. I do not see how Triton X-100 alone will lyse bacterial cells. (How much Triton X-100??).

2. Ranjan is right: If you are just testing if you are getting expression, use sonication to guarantee that your cells have lysed. 3-5 rounds of 20 sec sonication bursts with cooling on ice in between bursts should pop most of your cells. SONICATION GENERATES HEAT so always cool on ice in-between.

3. Don't forget to save the pellet and check that also after sonication.

4. Worry about His-tag only after you can confirm any expression. It looks like the buffer you used was for running the column??

5. Use Western blotting to confirm His-tag is present

Good luck !