Hello,

Arizaldos idea (boil lysis) is very nice if apliable as it will denature most other proteins and your protein will be rather pure.

Lysozyme (+DNase) or Bugbuster are other alternatives.

Freeze-thaw cycles will also disrupt the cells.

Hypotonic lysis is my current first choise (also express in BL21 DE3), it makes the outher membrane porous and proteins between outher and inner membranes are released. About 70% of the released protein is my target protein.

Good luck!

Use below handbook

Good lock

A French press (named after CHARLES STACY FRENCH, 1907-1995) works very nicely, if you can lay your hands on one. This instrument passes cells with high pressure through a very narrow opening (needle valve). Since the velocity of fluid is high at the centre of the opening, but 0 at the wall, the resulting shearing forces disrupt cells in the canal.

Hello, Juan Pablo,

If the recombinant proteins are not heat-labile, you may consider boil lysis method.

Best,

Arizaldo

Hello,

Arizaldos idea (boil lysis) is very nice if apliable as it will denature most other proteins and your protein will be rather pure.

Lysozyme (+DNase) or Bugbuster are other alternatives.

Freeze-thaw cycles will also disrupt the cells.

Hypotonic lysis is my current first choise (also express in BL21 DE3), it makes the outher membrane porous and proteins between outher and inner membranes are released. About 70% of the released protein is my target protein.

Good luck!

Hello Juan,

As long as your protein does not accumulate heavily in inclusion bodies and is soluble in your buffer, you could consider using Lysozyme.

To lyse your cells, first resuspend your cell pellet in 10 or 20 ml of your lysis buffer with 2 mM EDTA and add 1 mg/ml Lysozyme (preferably from a 10X stock in 20 mM Tris-HCl, pH 8.0 ; 2 mM EDTA; 1% Triton X-100). Please note, that the presence of EDTA in your buffers is required for Lysozyme activity on Gram-negative bacteria like E.coli.

Then, depending on the nature of your protein, shake the homogeneous suspension for 30 minutes to an hour at room temperature or up to 37°C.

After ultracentrifugation, you should obtain a clear, watery supernatant. If the solution is viscous or the pellet still contains considerable amounts of protein, you should vary the parameters for the incubation.

Hope this helps,

Best regards, Mario

I think you can use the same buffer which was used by you. You can use your buffer also but you might need to incubate for 1 h at least in ice or room temp. Better to use lysozyme if it is available. You can get enhanced lysis by freeze thaw in liquid nitrogen also.

Hope this will help you

with regards

shivjee

There are many other options. As mentioned above, you can use beadbeater using 0.5 mm beads. Zymo Research sells bacterial strains (BL21DE3 with inducible lysozyme gene). If you transform this strain with your DNA and induce lysozyme (arabinose-in my memory) and a few freeze-thaw cycles would do it.

An easy way to disrupt cells is a combination of hypotonic solutions and vigorous agitation in vortex equipment, after that you can continue with your standard protocol

You can use 8 M Urea solution and stirring for an hour. To make cell lysate for proteins.

You can use 8 M Urea solution and stirring for an hour. To make cell lysate for proteins.

I agree with Engelbert Buxbaum , the French press is the best option to me as this will not cause your protein to aggregate (which can happen when using chemical lysis). You can even parameter the press to use enough pressure to disrupt inclusion bodies. If you use the press, you don't need a specific buffer, only LB supplemented with protease inhibitors and nuclease. (> 25k psi)

If there is no French press available in your lab and you work with small volumes, you can use a very large syringe with the thinnest needle to reproduce the same process (you then have to repeat it several times). However, this will not give you the best yield and it is time consuming.