Engineering, design and selection of fluorescence-activating proteins for advanced imaging and biosensing

Abstract Fluorescence-activating proteins (FAP) have emerged as a novel class of genetically encoded tools for fluorescence-based protein imaging, complementing the existing toolkit consisting of fluorescent proteins and self-labeling tags. FAP have the ability to bind and activate the fluorescence of small molecules, called fluorogens, that are otherwise non-fluorescent, allowing protein localization with high specificity and little background. In this review, we present the engineering of FAP and FAP-based reporters from various protein scaffolds, focusing on the different strategies implemented to design and engineer their properties for specific biological imaging applications.