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E coli lysis by lysozyme


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#1 genz

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Posted 17 July 2010 - 03:36 AM

Hello everyone,

I am trying to lyse E coli with lysozyme. I have searched low and high, in most protocols it say use 1 mg/ml to lyse the bacteria. But when I went back to very old literature, most used 0.02 mg/ml of lysozyme to lyse bacteria.
I have tried with a different concentration and found interesting results:
1. E coli not treated with EDTA never lysed
2. E coli treated with EDTA lysed well from 0.02mg/ml till 1mg/ml (solution become translucent)
3. E coli treated with high concentration of lysozyme >2mg/ml not lysed... the solution actually turned very very cloudy, very strange
4. No matter how much lysozyme, the solution never become completely clear. For example, when transferring a small amount of the sample 2 above after lysis into a fresh solution of E coli cells, lysis occur but never complete (judge by transparency). Does it mean:
a) lysozyme are active but somehow don't lyse all the cells?
:lol: solution never come clear because the peptidoglycan layer only hydrolysed partially by lysozyme, other big molecules covalently linked but broken by lysozyme?
c) the LPS too large so they flow around in the solution and contribute to not 100% clear

In comparison with sonication, I always get very very clear lysate. Lysozme treatment can only best give me lysate ~50% clear compared to treatment with sonication.

Does anyone has a very good lysozyme treatment to lyse e coli 100%

Really appreciated

Genz

#2 msantos

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Posted 19 July 2010 - 09:10 AM

Hello, my name is Mary Ann Santos and I am a product scientist of InVitria. I would like to share with you the benefits of Lysobac, our animal free, recombinant human lysozyme that is used in the efficient lysing of E.coli cells. Below is a brief description of this product:

Lysobac™ is a breakthrough for bacterial cell lysis and can be used in diagnostic applications, bioprocessing and life science research. Lysobac is recombinant human lysozyme produced in an animal-free production system. Animal-free production eliminates the safety risk and inconsistent lot-to-lot performance of hen egg white lysozyme. Lysobac has significantly higher bioactivity than hen egg white lysozyme. One gram of Lysobac replaces 4 grams of hen egg white lysozyme. Lysobac also delivers gentle cell lysis, unlike mechanical lysis which can cause protein shear and reduce protein yields by up to 20%. When added to mechanical lysing, it can provide for a more complete lysing, increasing protein yields.

Advantages:

• Improves Bacterial Fermentation Efficiency
• Greater bacterial lysis activity per mg
• 1 gram of Lysobac delivers more lysis activity than 4 grams of HEWL
• Animal-free and consistent leading to improved regulatory efficiency for drug manufacturing
• Improved recombinant protein yield compared to mechanical lysis
• Reduces cost of goods

We work with groups that use Lysobac alone to lyse their E.coli. In these instances, they have reduced their usual hen lysozyme concentrations as described above and have resulted in complete lysing.

We also work with groups that combine Lysobac with sonication. One of the groups said that they regularly use hen lysozyme in combination with either freeze-thaw or sonication and they were able to reduce their lysozyme concentration 4 times and have resulted in a more complete lysing. Combining 0.1 mg/ml Lysobac with sonication have given the best results.

Lastly, the attached table shows the synergistic effect of Lysobac when used in combination with EDTA (concentrations are in micrograms/ml)

Please feel free to contact me at msantos@invitria.com if you would like to obtain a sample of Lysobac for evaluation or if you need any further information.

Thank you,

Mary Ann

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#3 msantos

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Posted 19 July 2010 - 10:14 AM

Hello again,

I just wanted to direct you to the web page below. You will also find a link to a journal article that may be of interest to you in this page (right hand side).

http://www.invitria....cell-lysis.html

I hope this helps and I hope that we can work together in the future.

Sincerely,
Mary Ann




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