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

Hongmin Cai,Hongmin Cai,Yanfang Li,Yanfang Li,Tingting Li,Tingting Li,Dianfan Li,Dianfan Li,Hebang Yao,Cedric A J Hutter,Markus A Seeger,Yannan Tang

doi:10.1038/s42003-020-01478-z

Abstract

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 °C (20-min heating). By contrast, the limit for the two popular GFPs is 64 °C and 74 °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.

Highlights

Green fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability
A more stable fluorescent protein for fluorescence-detection size exclusion chromatography (FSEC)-TS assays at high temperatures will be welcome
FSEC-TS assays for fluorescently tagged membrane proteins are best performed at temperatures where the tags are largely unaffected (~25% drop of fluorescence) so that the decrease of fluorescence can be largely attributed to the aggregation/precipitation of protein of interest (POI)

Summary

Introduction

Green fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability. 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. One limitation of the most commonly used GFPs, is their modest thermostability It fails to serve as a reporter for FSEC-TS of membrane proteins with apparent Tm exceeding the Tm of GFP itself which is ~76 °C (10-min heating)[46]. The overall superior characteristics of TGP over GFPs, together with the sybody tools, should encourage the use of TGP as a fusion partner for membrane proteins but may for soluble proteins

Methods

Results

Conclusion