I am currently working on a project where I need to recover and purify a number of His-tagged proteins for multiple 30 L fermentation runs. At smaller scales (around 1 L ferms) I have used a high pressure homogeniser (Constant Systems Ltd. CF1), but recently trying to lyse 2 L cell suspension by this method and it took a very long time!
What systems do others use for larger scale cell lysis? Any recommendations for equipment (available in the UK)?
Many thanks,
Joe
What kind of high-pressure homogenizer do you use?
This is the standard method to break bacterial cells; have used this method for large production scale; the largest one was a 5000L fermentation.
Give some details of the way you do it: pressure, temperature, ...
Hi Achim Recktenwald , I've been using a Constant Systems Ltd. continuous system (picture attached), that allows you to continually pour cell suspension in. I have been trying to keep the equipment at 10 oC or below (using some cooling water baths) and using 25 KPSI pressures.
I do not know this machine.Will look into this, when I have some time.
Your cooling has to be top-notch or your protein is cooked. All the energy is converted into heat. Assume an increase of 15C with 1000 bar.
What kind of bacteria are you trying to break?
With E. coli, I would use 800 - 1200 bar. You may have to test what is the best for your device. You can concentrate the bacteria up to 40% (v/v) wet cell mass. If your cooling is not good enough, go lower.
I would not pour the solution. The systems I know, all are able to suck the suspension from a container.
You may want to add some Benzonase (DNase, RNAse) to reduce the viscosity, which is generated by the release of DNA. I had once a system break down, because of the high viscosity, almost like jello. You do not need a lot; the enzyme is very effective.
What kind of impact plate has your system?
This is the surface onto which the suspension is shot, when it comes out of the high-pressure chamber. The cells take on a lot of water during the pressure phase to adjust the internal pressure in the cell. When they shoot out, they cannot release it fast enough and are shot like water-filled balloons onto the impact plate, which cuts them.
If one round is not sufficient to break the majority of your dells, send them in for another round or even two. But make suspension is sufficiently cooled down again.
The fewer rounds the better, as every round will the percentage of HCP released. I would not go beyond 3 rounds.
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