An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification

Green fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability. An ideal reporter should be stable itself and provide high sensitivity and yield. Here, we demonstrate that a coral (Galaxea fascicularis) thermostable GFP (TGP) is by such reasons an improved tag compared to the conventional jellyfish GFPs. TGP faithfully reports membrane protein stability at temperatures near 90 degrees C (20-min heating). By contrast, the limit for the two popular GFPs is 64 degrees C and 74 degrees C. Replacing GFPs with TGP increases yield for all four test membrane proteins in four expression systems. To establish TGP as an affinity tag for membrane protein purification, several high-affinity synthetic nanobodies (sybodies), including a non-competing pair, are generated, and the crystal structure of one complex is solved. Given these advantages, we anticipate that TGP becomes a widely used tool for membrane protein structural studies. In this work, the authors demonstrate that a coral thermostable GFP (TGP) is an improved tag compared to conventional GFPs. In addition to reporting melting point stability at temperatures near 90 degrees C, its fusion also helps increase expression levels of test membrane proteins. They further generated synthetic nanobodies against TGP to facilitate purification.