Superfolder Green Fluorescent Protein (sfGFP): A Versatile Tool for Biotechnology Research.

Superfolder green fluorescent protein (sfGFP) is an engineered GFP variant best known for its robust folding, reliable chromophore maturation, and high tolerance to fusion partners. This review argues that the principal value of sfGFP lies not in universal superiority in brightness or photostability, but in its ability to preserve fluorescence under conditions that compromise the folding or performance of conventional GFP variants. These contexts include difficult fusion proteins, split-fragment complementation systems, circularly permuted biosensor scaffolds, recombinant expression platforms, and secretion-associated workflows. Because sfGFP is directly encoded by a DNA sequence, it can be expressed in living cells as a fusion tag and generate fluorescence through autocatalytic chromophore maturation without requiring exogenous cofactors or synthetic fluorophores, enabling live-cell localization, protein detection, and dynamic biosensing. Its high solubility and folding robustness can also improve soluble recovery, reduce aggregation, and facilitate the purification of recombinant fusion proteins. However, sfGFP is not without limitations: its fluorescence remains pH-sensitive, its monomeric behavior can be context-dependent, and its photostability is not optimized for prolonged high-intensity illumination. Therefore, sfGFP should be selected according to application-specific requirements and benchmarked against newer green fluorescent proteins in terms of folding robustness, brightness, maturation kinetics, monomericity, pH tolerance, and photostability. Future integration of sfGFP with cell-free systems, synthetic biology, and protein-design strategies is likely to further establish it as a robust fluorescent scaffold for biotechnology.