GST-fusion protein purification from bacteria - Several questions on protein purification (Jul/22/2007 )

Hi folks,

I plan to purify GST fusion protein from bacteria (BL21) and have some questions on that.

1. After IPTG induction and before lyse the bacteria, can I freeze down the bacteria cell pellet at -80C and and lyse them later?

2. To lyse the bacteria by sonication, the protocol says 15s X 3 times.
A. At what strength (our lab sonicator has 0-10 selector) do I sonicate?
B. How long do I have to wait inbetween to cool down the solution?

Thank you!!!

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Hi,
1. It is OK, although of course fresh always has less chance of protein degradation
2. Actually, this depends a lot on experience and personal preference. I don't know if your machine is like ours, but ours has two color codes on the selector, black and red. the black ones are lower strength and the red ones are higher, thus more dangerous (for your ears). We normally use 4, right at the border between red and black. I did 3-4 times 15-20secs with 15-20secs rest in between. the main points are: do it in cold room, best is to keep your samples on ice, don't let samples heat up, don't put the sonicator tip too deeply inside the suspended bacteria, keep it right below the surface. You can check the sample from time to time, when it looks homogenous (like liquid, clearer than with just suspended bacteria, you can see the difference holding it up under the light and look through it), then it is ready. Can't describe better than this, sorry.

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1. After IPTG induction and before lyse the bacteria, can I freeze down the bacteria cell pellet at -80C and and lyse them later?: Freezing and thawing also lysed bacterial cells!


2. To lyse the bacteria by sonication, the protocol says 15s X 3 times. , you can also do 10s x 4. As Almasy pointed out, keep the samples on ice. Dont let the probe touch the wall of the tube.

A. At what strength (our lab sonicator has 0-10 selector) do I sonicate?-- you should choose the correct probe, which depends on your tube size. I often choose at 3 or 4, not too noisy.

B. How long do I have to wait inbetween to cool down the solution?--- just 1 min, or you should do various tubes, and take turn for each tube

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Hi guys

I have 3 GST-fusion proteins and I cannot get good expression after IPTG induction. The GST by itself is expressed very well. I was wondering if anyone knows how to improve the GST-fusion proteins expression from E-coli? Adding glucose will help? Or should I change the bacterial host?

Many thnx

Haki

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