Does anyone have any idea how to use affinity column to purify Avitagged protein? This protein is biotinylated in vivo by a biotin ligase? Can I bind it to Strepavidin beads and elute it with free biotin? I suspect binding of biotinylated to SA so strong and it won't elute. Anybody have an idea? Thanks
You should be able to elute the biotinylated protein from the streptavidin beads with free biotin, avoiding denaturing conditions, because the affinity of the free biotin-streptavidin interaction is stronger than the affinity of the interaction of streptavidin with biotin attached to something.
Dear Meral, thank you for your suggestion. I would avoid the denaturing conditions since I want to purify my protein in an active form.
Dear Adam. Thank you for your answer. That sounds a good option. Is there any literature that suggest the different binding affinity of free biotin to biotinylated protein? I wonder how much the difference is
The Kd of the streptavidin-biotin interaction is given as ~10-15 M, essentially irreversible. I haven't been able to find a value for the Kd of the interaction between a biotinylated protein and streptavidin, but the literature seems to indicate that harsh, probably denaturing conditions are needed. I observed during some experiments I did years ago that biotinylated oligonucleotides did not bind anywhere nearly as tightly to streptavidin as biotin does. The Kd was more like 10-8 M, still very tight, but not so tight as to make elution impossible. This is what leads me to think that it may be possible to elute biotinylated proteins using free biotin if (1) the biotin concentration is high enough and (2) enough time is allowed for the exchange of free biotin for slowly dissociating biotinylated protein.
Biotin solutions can be made at concentrations of at least 50 mM if the pH is neutralized. I think it would be worth trying to elute your protein with 50 mM biotin and an overnight incubation. If that doesn't work, then I think you should look for a different affinity tag if you want to recover native protein.
Purification of Biotinylated Enzymes Using Streptavidin Mutein Matrix
https://lifescience.roche.com/shop/products/purification-of-biotinylated-enzymes-using-streptavidin-mutein-matrix-biochemica-1-04-p22-24
The mutated streptavidin matrix with reduced biotin affinity (Kd ~ 10-7M), described in the paper to which Zhang Daniel drew our attention, was eluted with 2 mM biotin.
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