Lysis for Enzyme Purification

Open DNA Collections Enzyme Expression Protocols
1

Hey guys,
On the meeting we had a discussion on bacteria lysis. Some of you use the Thermo Fisher BPER Reagent, which is really expensive.

There are some much cheaper alternatives to that, I found a good summary with protocols on the EMBL page: https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_ecoli/

It says on the Thermo Fisher page, that this reagent is “Detergent in Tris buffer with Lysozyme and Universal Nuclease” and the safety sheets just show >=1 -<2.5% of Triton X-100 in it. I guess it should be easily exchangeable by home-brew recipies and protocols and you don’t need to buy it. pH of this reagent is 7.6.

An alternative protocol can be this one:
https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_ecoli/enzymatic_lysis/index.html
You can try adding Triton X-100 in the same concentration range and you can use AEBSF instead of PMSF, which is less toxic. Also the use of 1mM EDTA is for gram negative strains (like BL21) recommended (but not compatible with Ni2+ affinity purification or Mg2+ dependend enzymes like DNA polymerases! But you can also freeze them in PBS)

A good starting point to develop your own protocol is here:
https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/lysis_buffer_additives/

Have a good day!
Kathrin

2

Also the data sheet for lysosyme is interesting for protocol optimization:https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/7/l7651dat.pdf

3

And another mechanical procedure I’ve found:
Bead Beaters

This technique involves combining a cell sample with tiny beads in a bowl, which is then mechanically agitated in order to break the cells apart. To avoid overheating the sample, this should be done in a cold environment and the sample should be allowed to rest and cool between beating cycles.

4

Hi, I’m willing to test out this mechanical lysis protocol and I need to find a decent way to assess the results except or running acrylamide gel which are difficult to get.
Anybody has any idea? Maybe lawn culturing the before and after, plating and counting colonies?
I bought some glass beads to test it.

Cheers,
Adrian

1 Like
5

Hi Adrian,
Hm…your idea is to check for viable bacteria after lysis?
I would say just give it a try! Sounds like a very interesting idea.

Do you have a protocol suggestion for lyseing with glass beads? Would be glad to discuss it :slight_smile:

Cheers,
Kathrin