Recombinant Expression and Automated Flow Synthesis of Fold-Switching Proteins Derived from Streptococcus Protein G

Fold-switching proteins are an emerging class of biomolecules that respond to subtle cues to regulate biological processes. Expanding our understanding of fold-switching proteins requires reliable methods for preparing new variants with high purity and well-defined thermodynamic properties, but currently these methods remain limited. Here, we describe complementary methods for preparing fold-switching proteins by recombinant expression and, to our knowledge, the first reported total chemical synthesis. We prepared eight proteins derived from Streptococcus protein G: 56-residue GA and GB, with 95 and 98% sequence identity. Four variants were produced by bacterial expression in Escherichia coli as SUMO fusion proteins, followed by traceless on-column cleavage and size-exclusion chromatography to generate the native sequences. The same four variants were also produced by automated flow protein synthesis, followed by RP-HPLC and anion-exchange chromatography purification. Both workflows give highly pure proteins that adopt folded conformations upon chromatographic desalting into phosphate buffer. Circular dichroism shows that the proteins attain the anticipated 3−α or 4β+α topologies. Each pair of recombinant and synthetic proteins adopts a similar conformation, as corroborated by trypsin digestion and 1H NMR studies. Thermodynamic studies show that the melting temperatures are within 1 °C of each other and the energetic differences are less than ±0.1 kcal mol–1. Together, these methods provide benchmarks for dual chemical and biological preparations of fold-switching proteins, facilitating access to broader chemical space to study their structural and thermodynamic properties and to design new proteins with switchable properties for applications in science and medicine.