Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology

Abstract

Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein–protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology.

Biophysical Techniques for Target Validation and Drug Discovery in Transcription-Targeted Therapy

“…Assays using fluorescence or luminescence as read-outs have been particularly useful in drug discovery as these signals can be easily quantified, especially in a high-throughput format. Recombination of a fluorescent protein [87] or luciferase and its derivatives [88] is used in bimolecular fluorescence complementation (BiFC) [89] or bimolecular luminescence complementation (BiLC) [90] assays. BiFC and BiLC have been used to detect PPIs in a variety of organisms, from yeast [91] to plants [92].…”

Substitutional landscape of a split fluorescent protein fragment using high-density peptide microarrays

“…Ideally, left-out elements can spontaneously associate with the LOO-FP to recover fluorescence, making them useful as tags fused to a protein of interest, as well as individual peptides for in vitro applications. Preferably, a tag should have high solubility, affinity and brightness upon complementation of the LOO fragment, as well as a small size that interferes minimally with the fusion partner protein (2,5,7,8). Amino-acid substitutions in the sequence of the LOO-FP fragments have the potential to modulate their complementation efficiency, spectral properties, solubility and photostability.…”

A Novel Split Reporter Uncages New Possibilities

“…Yeast two-hybrid screening, the most widely used PPI screening method, uses a split-transcription factor as a reporter and detects the transcription of a specific gene. Bimolecular fluorescence or luminescence complementation assays (BiFCs and BiLCs, respectively) rely on the recombination of a fluorescent protein 2 or luciferase and its derivatives 3 upon interaction between two proteins of interest. A variety of enzymes have been used as scaffolds for split reporters, providing a variety of read-outs, ranging from colorimetric tests as in the case of the split-β-galactosidase to cell viability (split-β-lactamase).…”

“…Fluorescence, for example, is one of the most used read-outs in cellular biology as it gives information on the localization of the protein within the cell. Multiple groups have fine-tuned BiFC to provide subcellular localization, 2 including organelle-specific targeting of proteins, or even allow subdiffraction localization to be obtained. However, the use of green fluorescent protein and its derivatives is poorly amenable to in vivo imaging where autofluorescence and light penetration are limiting factors.…”

“…This is the case for the cyan fluorescent protein that can be reconstituted by its own fragment and emit light at 480 nm, or it can bind to a fragment of the yellow fluorescent protein to produce a fluorophore that emits at 530 nm. 2 Conversely, the fragment of the click beetle luciferase (green) can recombine either with its own scaffold and emit green light (540 nm) or with the scaffold of the click beetle luciferase (red) and emit luminescence in the red region (615 nm). 5 Competition between proteins for binding to a common interactor can be measured by simply comparing fluorescence/luminescence in two detection channels.…”