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

• Published in: Communications biology Vol. 3; no. 1; p. 753
• Main Authors: Cai, Hongmin, Yao, Hebang, Li, Tingting, Hutter, Cedric A J, Li, Yanfang, Tang, Yannan, Seeger, Markus A, Li, Dianfan
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 10.12.2020; Nature Publishing Group UK; Nature Portfolio
• Subjects: Affinity; Biology; Chromatography, Affinity; Crystal structure; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genes, Reporter; Green fluorescent protein; Green Fluorescent Proteins - chemistry; Luminescent Proteins; Membrane proteins; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - isolation & purification; Models, Molecular; Nanobodies; Protein Conformation; Protein expression; Protein purification; Protein Stability; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Single-Domain Antibodies - chemistry
• ISSN: 2399-3642; 2399-3642
• DOI: 10.1038/s42003-020-01478-z

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.