Lyse Bacterial Pellets Expressing Human Recombinant G4 Resolvase1 (rG4R1)
3:06
Prepare G4-magnetic Beads
4:07
Bind Histidine-tagged rG4R1 to Cobalt Beads
5:47
Elute rG4R1 from Cobalt Beads
7:07
Bind rG4R1 to G4-bound SPB and Elute in an ATP-dependent Manner
8:55
Results: Analysis and Quantification of Catalytically Active, Purified rG4R1
10:06
Conclusion
Transcript
The overall goal of this method is to isolate the G-quadruplex helicase G4 Resolvase1 protein from a bacterial expression system using a unique ATP dependent purification step to isolate nearly pure and catalytically active enzyme. This method can
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G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.
