I want to know how effective is the enzymatic lysis for recombinant protein extraction from E. coli.
I saw in Sambrook and Russell their protocol. However, here at my departament we dont use it alone. We put 1mg/ml of Lysozime, incubate 30 minutes in ice and after we proceed to sonication.
I've asked why we do that, because the protocol of Sambrook and Russell doesn't and it is far more easy and reproduceable.
Some of you are using a enzymatic cell lysis protocol for protein extraction as sole lysis protocol?
Thank you in advance
Lysozyme only digests the outer wall of the bacteria. It does not disrupt the cell membrane. Lysozyme digestion is used to make spheroplasts, which can fused onto cells to transfect bacterial plasmids. In order to get the cytoplasmic proteins released, you have to disrupt the membrane. This can be done by sonication, pressure cell or by detergents such as deoxycholate. SDS is not recommended for protein isolation, because it denatures protein.
It is better to disrupt the cell by High Pressure homogenisation, French press to release the intracellular proteins. SDS or other chemical may denature your proteins.
Hope it will work.
Good luck
As Gary says lysozyme is always accompanied by a detergent in lysis buffers. Yet, sonication is needed if you don't use a French Press, as also suggested, to fragment the bacterial genomic DNA and the protein aggregates that inevitably form if you have a high expression level of your protein, even if it is properly folded and soluble. Obviously sonication is a critical step, the power and the timing has to be carefully regulated, because it warms up the lysate and if temperature rises too much it will denature your protein, making the native lysis approach useless!
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