Hi,
I have produced a 37kda viral protein using Baculovirus expression system (FlashBac Ultra)/insect cells. It is a glycosylated protein and contains a transmembrane region at N-terminal. Protein also has a 6-his tag at C-terminal. I got the protein band in SDS PAGE and WB using cell pellet. No protein was detected in the supernatant. I also did MOI and time of harvest experiments and found that MOI 5 at 72 h gives a better expression. Now, I need to purify the protein from the cell pellet. I will be using HisPur Cobalt Purification Kit from Thermo. Can anyone suggest how to solubilize the protein present in the cell pellet for his-tag purification? I did some literature search and found that using either 8M Urea of Guanidine can be useful.
Thank you
Deepak
You may try 8M urea or you will have to try different detergents and find out if any detergent is more useful to solubilize your protein.
Hi there,
Your protein is insoluble because it is an integral protein. You still may have a go with native purification (ie. without urea or GdmCl) but you will have to include a detergent to solubilize the membrane fraction. In my experience 2% DDM is a good starting point and it is fully compatible with IMAC.
Thanks Mahesh and Jiang. Using urea or any other detergent will unfold the protein and refolding the protein might also result in loss of protein? I am using insect cells and expression is not so great. I need to use this protein for standardizing indirect ELISA to detect antibodies. Are you aware of any methods which do not use strong detergents?
Thanks Dominique. So I just need to add 2% DDM in the lysis buffer? Do you have a protocol?
Hi again, once you have your membrane pellet (ie. after cell lysis and centrifugation) you have to resuspend it in a DDM containing buffer. 2% is the usual concentration used for extraction then DDM is decreased during the purification steps (down to 0.1% in my own experience) or even exchanged for an other detergent. I think the following paper should help you: Article Expression, Detergent Solubilization, and Purification of a ...
I will try DDM, DM, OG. All methods do use detergents, DDM is first choice for many folks.If you want less detergent then once you extract protein, you may dilute the stock such that it is 2-3 times cmc or less.
Hi Deepak, any chance that the protein can be targeted to the membrane? You may want to try extracting the protein from the purified cell membrane (if it's present in the membrane) using DDM. Check this out for more ideas:
Article Mammalian Membrane Protein Expression in Baculovirus-Infecte...
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