Hello,
I am trying to purify a mammalian enzyme from bacteria E.coli (BL21(DE3)) and I have cloned the gene into pet28a and start to do induction with IPTG. After lysis with Lysozyme containing buffer (tris, glucose, EDTA and lysozyme) I can see that I am able to induce it. (Lane 6 in ponceau stained membrane). But the problem with this is I cannot get clear lysate. Which means I cannot purify it with Ni-NTA columns. When I do centrifugation and get supernatant I don't have my protein. Which I think it is in inclusion body and when I boil it I can take it out... When I do 8M urea containing lysis (urea, NaCl and KH2PO4) to open inclusion body I cannot get the whole induction again but at least I get clear lysate containing my protein, means when I do centrifugation my protein is in the supernatant. (Lane 4 after dialysis with same buffer without urea). Then I thought now the lysis is not good that's why I got less protein. So I was thinking of adding both of urea and Lysozyme together to have both good lysis and good opening of inclusion bodies. Do you think it is ok to use both of them together or do you think 6M guanidine HCl is better for my situation because of the enzymatic activity beside of the fact that mammalian post-translational modification problems etc..
It will be very nice If I can find someone to help me with this, which I cannot come up with a solution.
Thank you in advance,
Sinem
The first thing you should do is see if you can identify induction conditions under which at least some of the protein is in the soluble fraction without using urea. This can sometimes be achieved by cooling the culture down to room temperature or 16oC before inducing it, then continuing to grow the culture at the lower temperature. The slower rate of protein production sometimes allows the protein to fold properly and stay soluble instead of forming inclusion bodies. Using a different expression strain can also improve solubility. Working with urea-solubilized inclusion bodies results in having to find conditions to refold the protein, which should be your last resort.
I actually did induction at 28oC thinking that I may get rid of inclusion bodies, but 16oC I have never tried..
I know urea is risky but..
For 16oC situation, I will grow pre-culture at 37oC and after o/n incubation I will put some to main culture and grow this main culture at 37oC until O.D. 0.6 and do induction at 16oC, If I didn't misunderstand you, right ?
Thank you so much!
Putting urea and lysozyme in the same buffer is going to denature lysozyme. So the lysis will not occur. Now the deal is that firstly lyse the cells and centrifuge to collect the pellet. The pellet can then be resolublized in urea containing buffer. Once you get soluble protein, then dialysis out the urea. The dialysis buffer has to be such that it allows refolding of your protein . There are well established and published protocols for this.
Anyway, you are getting good overexpression of the protein.
Good luck.
Hi,
I agree with the suggestions given by Mohammad Asif Shah to first lyse the cells in the presence of lysozyme and then centrifuge the lysate to collect the inclusion bodies which can be washed with 8M urea buffer for solubilization. Alternatively, you can incubate the cells with lysozme for 1h and then go for sonication for 20 or 30 cycles by keeping the lysates in ice to avoid damage to the protein. This method can increase the efficiency of the lysis. After this you can go for centrifugation. If you find your protein in the pellet i.e. inclusion bodies then you have to solubilize it with 8M urea buffer.
So do you also have knowledge about 6M guanidine HCl ?
Thank you so much !
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