FLEX: A heparin-binding fusion partner engineered from fibroblast growth factor 1 to enhance protein expression, solubility and purity

Abstract

Expression of aggregation-prone, unstable, or cytotoxic recombinant proteins remains a major bottleneck in both academic and industrial research. Although solubility-enhancing affinity tags can improve expression, they often compromise purification stringency, increase construct size, or require additional downstream processing. Here we report FLEX, a compact 15.5-kDa dual-function fusion tag engineered from human fibroblast growth factor-1 (FGF1) that integrates intrinsic protein-stabilising properties with high-affinity heparin binding. Structure-guided computational redesign of the FGF1 scaffold reduced exposed hydrophobic residues, removed flexible protease-susceptible regions, and expanded the electropositive surface while preserving the canonical heparin-binding interface. FLEX exhibits markedly improved thermal and chemical stability relative to wild-type FGF1 together with enhanced heparin affinity, enabling high-stringency washing and improved purity in a single affinity step. We demonstrate the broad utility of FLEX by expressing and purifying a panel of challenging proteins in Escherichia coli, including cytotoxic Pseudomonas aeruginosa virulence factors that are difficult to obtain in active form. Unexpectedly, FLEX also performed robustly in mammalian expression systems, where transiently expressed FLEX-tagged proteins were recovered at higher yield and purity than with gold standard Myc and Strep tags, including difficult targets such as Tribbles 3 (TRIB3). These findings establish FLEX as a versatile affinity-and-stabilisation tag that improves expression and purification across diverse systems, providing a practical new tool for structural, biochemical, and translational studies of otherwise intractable proteins.