I am trying to purify an integral membrane protein of viral origin, and this protein goes to the ER membrane when virus infects the cells. However, for it to insert in the ER membrane, it needs a 23 AA signal sequence. I have made a construct wherein the N-terminal of this protein has been tagged with 11XHis (There is no signal sequence, however). I can see appreciable (not great) expression levels in SDS gels (with small cultures), however, when I do a large scale purification and pass the processed lysate through Ni-column, I lose all my protein. They don't properly bind to the Ni-mat and everything comes in the flow through. There are few questions I would like to ask:
a. Is it necessary for me to add the signal sequence as well so that the protein gets inserted in E coli membrane?
b. What is probably going wrong with the His-tagged protein not binding properly to the Ni-matrix? what are different methods can we try to express and purify these types of protein that might have worked for other people?
Hi there,
So your viral protein is inserted in membrane, right? So what is the topology of this protein in the ER membrane (is its Nter in the lumen or in the cytosol?)? If Nter is in the lumen, it absolutely need signal sequence at the Nter to initiate translocation. If the Nter is cytosolic the protein sequence should contain all the signal for its proper insertion in the membrane According to what you say ("it needs a 23aa signal sequence"), Nter should be in the lumen and therefore expression without this signal sequence has no chance to succeed (see the attached pictures for a better view on the situation)
What is going wrong in your expression and purification experiment is that your membrane protein cannot insert into the ER membrane and then is certainly not properly folded either. So I would suggest you to fuse your gene with a proper signal sequence at Nter and maybe place the His tag at the Cter to avoid any interference with translocation process. As a result you will expect the overexpression of a membrane protein therefore you will have to consider the presence of non denaturing detergent for solubilisation and purification (DDM is the most used non ionic detergent)
Hi Dominique,
Thank you very much for your answer. However, I would like to clear few things about topology and expression. It is an integral membrane protein with its N terminal side in the ER lumen and c-terminal in the cytosol. The signal sequence also happens to be the portion of another membrane protein preceding it.
However, I am performing the expression in the bacterial system (E. coli). Do I still need the signal sequence?
Hi Greta,
The proper translocation of protein does require the signal sequence unless you are trying to make it soluble. Dominique has better explanation over the issue. You should include the signal sequence to translocate the protein to its actual location and then try to purify it. In our case, we remove n-terminal signal sequence and c-terminal anchor regions to bring the protein into cytosol, thereafter purification is initiated.
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