Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

Green fluorescent protein (GFP) is one of the most widely used tools in life sciences. To capitalize on its ubiquitous usage, we developed two distinct designed ankyrin repeat proteins (DARPins) able to simultaneously bind to GFP for a publication in the journal Scientific Reports. We accomplished this by mutating and re-designing the clashing ends of the binding DARPins, based on information gathered from X-ray crystallography, with the software suite Rosetta.

The tools developed in this project can then be used to couple other modalities to the GFP-binding DARPins, in order to co-localize them in living cells. Furthermore, we linked two DARPins binding to the distinct binding sites on GFP into one protein. The resulting DARPin clamp bound GFP with a substantially higher affinity and can for instance be used in situations with very weakly expressed GFP. Overall, this was one of my first forays into pure protein engineering, yet still rooted in my original background of structural biology, and was very exciting to me!

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