Lactobacillus crude protein extraction? - (Apr/14/2011 )

Hi

I'm wondering if anyone has a basic method of extracting total protein from Lactobacillus?

Within the lab i work they use "BugBuster" but im told this only works for Gram Neg organisms.

I have tried resuspending in Ringers and lysing using 212um glass beads and a MagnaLyser (3000rpm 45sec), but i fear this is shredding everything! (I usually work with DNA/RNA so apologies if this seems barbaric.)
Nothing comes up on an SDS gel anyway.

Is it as simple as adding lysozyme??..and wouldnt this intefere/show up on SDS gel?

Thanks for your help in advance.

Chris22

The real question is what methods are you using to analyze your lysate? Many methods for lysing cells can introduce compounds that will interfere with downstream methods. If you are just interested in lysing the cells to collect total protein try 2% SDS with 25mM Ammonium bicarbonate. It is a pretty harsh lysis buffer but it will solubilize just about anything you put in it. This should work if you want to perform SDS-PAGE, if you want to do something else with the lysate there are other options out there.

proteaMatt on Thu Apr 14 13:24:55 2011 said:

Hi proteaMatt

Thanks for replying. The reason i am asking is i am over-expressing a recombinant protein within a lactobacillus host.
I have RT-PCR cDNA gene expression work completed on it, but just want to run an SDS-PAGE (and Agilent Bioanalyser) gel to confirm the protein size, and the difference between an induced and uninduced expression state from total protein extraction.

So that would be the endpoint for me.

I will try the SDS/Ammonium bicarbonate lysis you mentioned. Do you possibly have a protocol for this, or is it pretty slapdash, mix with cells, wait, load on gel?

Thanks again.

Chris22

Here is the protocol for cell lysis that I normally follow. The lysis buffer I recommended for you is different from the one in the protocol I linked, but this protocol will work for a SDS/AmBiC lysis buffer.

What strain you are working with?
Instead of using lysozyme i would use the muralytic enzyme mutanolysin ...this works good for lactobacilli. For sure you will find your protein that you used for lysis on your SDS-PAGE gel ...but you knwo the size of those protein so you don't have to worry.

Also keep in mind that some of the Lactobacilli have an S-layer and therefore are hard to crack. With those strains mutanolysin and SDS does not work ...you need to use more harsh conditions stripping of the S-layer by using NaOH or LiCl.

Good luck!
Regards,
p

Hi proteaMatt

Thanks for the protocol, its good to have it as a foundation, i can substitute in the enzymes and mechanical lysis steps as i need them.

pDNA on Sat Apr 16 07:25:51 2011 said:

Hi pDNA
It is a L.salivarius strain, when isolating plasmid DNA from this strain i actually used a combination of Lyzozyme and mutanolysin, but i was unaware if these may degrade proteins so i was reluctant to use them.

And funnily enough when electro-transforming this strain i had (still have) issues relating to its efficiency, and used NaOH and Lithium Acetate to try to improve this. I will try to apply these to the protein extraction also. Thanks for the suggestions.

On a side note, any suggestions to improve electroporation protocols for difficult lactobacillus strains would be greatly appreciated! Thanks again for the help!!

Chris22

Dear chris,

lysozyme and mutanolysin do not degrade proteins since they are glycoside hydrolases, so don't worry.

Have you ever tried pre-incubation of your cells in medium containing high concentrations of glycine (e.g. MRS) to improve electrocompetency?

Regards,
p

Chris22 on Sun Apr 17 13:33:24 2011 said:

Hi pDNA

Its good to know that lysozyme and mutanolysin wont degrade the proteins, im running fresh gels tomorrow, fingers crossed! Thanks for that.

For the electrocompetency protocols i have attempted a number of weird and wonderful techniques to try to improve efficiency, with only some success but not at all reproducible.

I have tried Glycine as you mentioned, at varying concentrations, both overnight and using Glycine shock for 1hour before harvesting.

I have also tried Penicillin G (usually at approx 6-12 microgram/ml) and tried DL-threonine in the MRS growth medium as cell wall weakeners. (seperate experiments)

A recent paper suggested growth in high concentrations of NaCL, which worked at an efficiency of 2 colonies/ug plasmid DNA. (Once)

Unfortunately my current project requires transformation of a bank of 20 genes into the Lactobacillus strain. So my work is cut out for me.

But if i have missed any method id be more than happy to try any crazy ones out!

Thankyou for the input.

Chris22

I was talking with one of our analytical chemists and we were both amazed that a 2% SDS solution wouldn't lyse those cells. I was just curious if you tried it and what your results were.