Premature cell lysis - (Mar/15/2011 )

Hello,

I've been using BL21(DE3)pLysS cells for a number of different transformation and expression projects as a beginning step in protein purification. Lately, I've been growing larger cultures and harvesting the cells, but I've been having problems with premature cell lysis. Normally, I centrifuge 6L of culture, then resuspend in TBS or a similar buffer, centrifuge again, then store the pellet. I've been experiencing a large degree of cell lysis during resuspension. If I aliquot my resuspended cells into 50mL tubes, after centrifugation, approximately 25mL of the 50mL is the snotty lysis mixture (presumably DNA). If this is eliminated, I am only left with a very small pellet, about half the size (or less of what I would expect). I am wondering:

1. If I eliminate the DNA (say, decant the DNA and keep what of the pellet is left), will I even have any protein to work with in this pellet, or was it all eliminated with the DNA mixture?

and

2. What can I do to reduce this problem? I know that I should be using cold buffer; however, in using the same cell line to purify other proteins in the past, I have not experienced this much lysis during resuspension. I do keep the pellets on ice during the procedure.

Thanks for any advice.

Hi, I read your post and I think that you could recovered proteins more purified fron DNA using phases. So if you do a phase clorophormo-metanol-water you find proteins in aquos phase.
Please let me know if you decide to perform this method the obtained result
thank you

Hola In my experience lysis occurs sometimes about 4h post induction at 37ºC it depends of inducer, recombinant protein etc. If your next step after resuspension is sonication or french press, DNA will be broken and the solution will lost viscosity. Other practical way to avoid lysis is induce at 25-28ºC and collect 30 min before. Buena suerte