Hi
I am going to try E.Coli Rosetta 2 (DE3) pLyss strain for protein expression. I have read some papers and found pLys strain does not need sonication for lysing because of Lysozyme expression. My target protein is very sensitive to heat and I don't want to use sonication. What is the good way to lyse the E.Coli using expressed Lysozyme. Simply freeze/thaw e.coli and stirring at low temp?
Yes. Use lysis buffer with EDTA & non-ionic detergents. Do a single freeze-thaw cycle. Works like a charm.
Except for the "stir at low temp" part. I think lysozyme needs to come to a reasonable temperature to work well. I haven't needed to optimize this directly, but it's pretty clear to see by eye that nothing much happens until the last ice is melted and it feels warm to the touch.
Yes. Use lysis buffer with EDTA & non-ionic detergents. Do a single freeze-thaw cycle. Works like a charm.
Except for the "stir at low temp" part. I think lysozyme needs to come to a reasonable temperature to work well. I haven't needed to optimize this directly, but it's pretty clear to see by eye that nothing much happens until the last ice is melted and it feels warm to the touch.
Thank you John and Chara. I need some more advice. When I sonicate the E. Coli, I store only pellet of bacteria and stored at -80 degree then add lysis buffer to the frozen bacteria and sonicate. When using lysozyme, Should I suspend the E. Coli with buffer such as PBS before put it into refrigerator?
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