Protein insolubility problem after bacterial induction - (Oct/06/2008 )
I have expressed my proteins in the pET42a vector and got induction after the addition of IPTG in Rosetta DE3 bacterial strain. The proteins are tagged with 3 tags: GST, 6-His and S-tags (which all can be removed afterwards). In my initial trials I had used loading dye to lyse the cells and loaded directly on the gel to see induction. In order to purify, we chose to use Ni-NTA columns firstly and I used 8M urea or 6M guanidine hydrochloride pH=8 supplemented with 0,1M NaH2PO4 and 0,01M Tris.Cl. This is the recommended buffer recipe directly from Qiagen's handbook. Yet, both of my proteins came down in the pellet in the insoluble fraction even before I loaded on the column! To solve the problem I tried inducing the protein at lower temperatures (13oC, 16oC, and 20oC overnight) and/or lysing in the initially recommended buffer plus NaCl (500mM), BME (20mM), Glycerol (10%), NP40 (0,1%), SDS (0.1%), Tween 80 (0,1%), Ethanol (10%) one by one or all together and none of these worked out. The proteins are hydrophobic. Could you please make any other suggestions to solubilize the protein?
Thanks Thanks Thanks
As i understand u used buffer 8M Urea for solubilize ur protein and yet it didnt appeared in the sup.
its not make much sense because this kind of buffer should be the choice for insoluble proteins (i.e inclusion bodies).
i think u should repeat ur test by W.B analysis for protein verification. maybe the protein that u see and think for ur protein by
commasie staining, is not the right one.
Another approach is using Inclusion body purification protocol- first u separate all the soluble and undesired proteins from the pellet
(with native lysis buffer and with PBS washing) then u solubilize the unsoluble proteins with UREA or GuHCl from the pellet.
Thanks for the answer. I am pretty convinced the protein is right, since there is no corresponding band in the -IPTG samples and a giant band in the +IPTG samples at the expected size. I have come across other people having similar problems using 8M urea in other polls and inclusion body purification is recommended like you suggested followed by grinding the protein in liquid N2. Do you have a suggestion for a protocol to purify inclusion bodies? I was not doing such a separation.
Once again thanks.