Applications of fluorescent protein tagging in structural studies of membrane proteins

Generating active, pure, and monodisperse protein remains a major bottleneck for structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM). The current methodology heavily relies on overexpressing the recombinant protein fused with a histidine tag in conventional expression systems and evaluating the quality and stability of purified protein using size exclusion chromatography (SEC). This requires a large amount of protein and can be highly laborious and time consuming. Therefore, this approach is not suitable for high-throughput screening and low-expressing macromolecules, particularly eukaryotic membrane proteins. Using fluorescent proteins fused to the target protein (applicable to both soluble and membrane proteins) enables rapid and efficient screening of expression level and monodispersity of tens of unpurified constructs using fluorescence-based size exclusion chromatography (FSEC). Moreover, FSEC proves valuable for screening multiple detergents to identify the most stabilizing agent in the case of membrane proteins. Additionally, FSEC can facilitate nanodisc reconstitution by determining the optimal ratio of membrane scaffold protein (MSP), lipids, and target protein. The distinct advantages offered by FSEC indicate that fluorescent proteins can serve as a viable alternative to commonly used affinity tags for both characterization and purification purposes. In this review, I will summarize the advantages of this technique using examples from my own work. It should be noted that this article is not intended to provide an exhaustive review of all available literature, but rather to offer representative examples of FSEC applications.