Hi everybody,
I created a recombinant protein consisting of a FLAG tag, my protein of interest and a Rat Fc (IgG2a: Ch1, hinge, Ch2, Ch3). Total size is approximately 60 kDa and the protein is supposed to be soluble and secreted.
When I try to purify the protein with FPLC by running the cell culture medium over an Akta Hi-Trap protein G column, there is barely any protein in the purified fraction and almost all protein goes straight to the flowthrough. (compared the amount of protein of interest in purified fraction with flowthrough by ELISA).
The Rat-Fc is present because I can detect the protein by WB, ELISA and FACS using anti-Rat secondary Ab's. Also the size estimation by WB implies that the Rat Fc is not missing (although the protein runs 2-3 KDa higher than I expect).
I also tried purification by protein A/G agarose beads and a different column to exclude problems with the particular column I used. I use a phosphate buffer (pH 7.0) as a binding buffer and I directly apply filtered cell culture medium (adjusted to pH 7.0) to the column.
Basically my question is whether I should be worried about the funtionality of the Fc and how it can be that the protein is not bound by protein G.
Thanks in advance,
Niels Dammes
Rat IgG bind very badly to protein A and G. The only exception is isotype IgG2a with protein G .... so this is probably not your problem. Protein G binds albumin very well (and I guess that you have FBS in your culture medium), and this could compete out your IgG. Most recombinant protein G are engineered to minimise this, but it might be wise to check the data sheet. Finally, did you do wb, ELISA and FACS with whole cell extracts or permeabilized cells ? In other words, are you sure that your Fc-tagged protein is secreted ?
Thanks for your reply. I transfect in serum free medium so I think that the albumin is not a problem. I produce the protein in Expi293F which are optimized for a specific serum free medium.
When I do my analysis I test both culture medium and cell extracts. It is secreted even though there is also a lare amount of protein still in the cells. But the amounts are anyway very high so there is more than enough protein in the medium.
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