I am studying a single-chain variable fragment (scFv) linked to a luciferase (Nanoluc) at the C-terminus (N-scFv-linker-Nluc-C).
The naked scFv (not linked to Nluc) can be easily purified using a Protein L affinity column (PL binds to the scFv), so I tried to use the same method to purify the fusion protein (scFv-Nluc). Nothing bound to the column. This is very weird since I used Protein L-HRP to perform ELISAs on scFv-Nluc and it worked (the signal was weaker compared to the naked scFv, but it was still a strong signal).
The difference between ELISA and purification is the time that the protein is "incubated" with Protein L. In ELISA, the incubation time was 1 hour. The purification was carried out with a flow of 1 mL/min (1 mL-column).
Can anyone explain this?
Is it possible that the Nluc-fusion disturbs the interaction with protein L and it "needs more time" to be found by protein L compared to the naked scFv?
It could be the time, or perhaps something funky about how the protein L is linked to the bead.
You could probe that. Pre-incubate the fusion protein with the purification resin, for 1h with very mild shaking (not to grind up the beads). Then, you can do two things: Pack the column in binding buffer, wash and elute normally. And use your elisa reagents to see if you can detect the fusion bound to the beads, using the normal elisa protocol.
Try changing the order of the proteins and add it to the C-terminal; change the linker or even try without a linker.
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