protein in inclusion bodies-plz help - (Dec/30/2008 )

Hello

I am making a GST-tagged fusion protein but facing serious problems:

i could get the desired GST-tagged protein ( the expected size) but my protein is in the inclusion bodies. To overcome this problem, I tried to solubulize the protein first : 7.0M urea worked efficiently to take out the protein in the soluble fraction but however this didnt bind the beads. So, I tried to dilute the urea concentration to 2.0M and check the beads binding but it also didnt work.

Previously I also tried to reduce IPTG induction time/concentration but protein remains in the insoluble fraction.

Finally I tried to use N-lauryl sarcosine in combination of 2% Triton-X100 according to the paper by Frangioni JV and Neel BG, 1993. This method could take out the protein in soluble fraction but this time also it didnt bind the beads.

I am now trying to reduce the temperature after induction and don't know whether it would work or not.

Does anybody has a better suggestion to solve this problem? I would appreciate very much if you have any advice.

There are kits that I checked in the internet that they are claiming to solve inclusion bodies as well as binding problem but I am afraid we couldnt buy it as we have limited funds in the lab.


plz help me

regards

hasina

Hi

I had a similar problem and I got around it by not using affinity purification at all. I increased the urea concentration stepwise from 1, 2, 3 M and so on till 8 M and ran each fraction on gel. What I observed was that most of my protein dissolved in about 5 M Urea. So I washed away all contaminating proteins with 4M urea and resuspended the pellet in a very small volume of 5M Urea. This had my target protein at very high concentration and the protein was relatively pure. I snap folded this in buffer (100 times dilution) and even after dilution the concentration was good enough to use the protein directly.

I know it sounds weird but it worked for me.

If nothing works, maybe you can give it a shot

TC

Hi

I had a similar problem and I got around it by not using affinity purification at all. I increased the urea concentration stepwise from 1, 2, 3 M and so on till 8 M and ran each fraction on gel. What I observed was that most of my protein dissolved in about 5 M Urea. So I washed away all contaminating proteins with 4M urea and resuspended the pellet in a very small volume of 5M Urea. This had my target protein at very high concentration and the protein was relatively pure. I snap folded this in buffer (100 times dilution) and even after dilution the concentration was good enough to use the protein directly.

I know it sounds weird but it worked for me.

If nothing works, maybe you can give it a shot

TC

Nope

I was very unlucky, it still didn't bind for me. In one case urea didn't really matter so i used the 100X diluted protein directly. I another case, I loaded it on resource Q, strong anion exchanger on FPLC and purified it.

You can check binding, if it binds. And make sure everything is chilled (including beakers and all) when you snap refold as the temperature is critical. Don't keep protein in 5M urea on ice, urea will precipitate.

TC

QUOTE (tarun @ Dec 30 2008, 09:14 AM)

Hi

I had a similar problem and I got around it by not using affinity purification at all. I increased the urea concentration stepwise from 1, 2, 3 M and so on till 8 M and ran each fraction on gel. What I observed was that most of my protein dissolved in about 5 M Urea. So I washed away all contaminating proteins with 4M urea and resuspended the pellet in a very small volume of 5M Urea. This had my target protein at very high concentration and the protein was relatively pure. I snap folded this in buffer (100 times dilution) and even after dilution the concentration was good enough to use the protein directly.

I know it sounds weird but it worked for me.

If nothing works, maybe you can give it a shot

TC

Dear TC

thx very much for ur mail.

u mean that after resuspending the pellet in 5M urea, u then dilute the urea concentration 100x and then check beads binding?

thank you.