Recombinant Expression and Automated Flow Synthesis of Fold-Switching Proteins Derived from Streptococcus Protein G
Clara Kjerfve1, Joanna Koryo Kwao1, Jordan Wolfe1
1Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, South Carolina 29208, United States.
Biochemistry
|April 1, 2026
Summary
Researchers developed new methods for preparing fold-switching proteins using both recombinant expression and chemical synthesis. These complementary techniques yield highly pure proteins with consistent thermodynamic properties, advancing the study of these biomolecules.
Area of Science:
- Biochemistry
- Protein Engineering
- Chemical Biology
Background:
- Fold-switching proteins are crucial regulators of biological processes.
- Current methods for preparing pure fold-switching proteins with defined thermodynamic properties are limited.
- Understanding these proteins requires reliable and accessible preparation techniques.
Purpose of the Study:
- To develop and validate complementary methods for preparing fold-switching proteins.
- To establish reliable recombinant and total chemical synthesis workflows for fold-switching proteins.
- To enable broader exploration of fold-switching protein chemical space for research and applications.
Main Methods:
- Recombinant expression in *Escherichia coli* as SUMO fusions, followed by on-column cleavage and size-exclusion chromatography.
- Automated flow protein synthesis followed by RP-HPLC and anion-exchange chromatography.
- Characterization using circular dichroism, trypsin digestion, 1H NMR, and thermodynamic studies.
Main Results:
- Both recombinant and chemical synthesis methods yielded highly pure fold-switching proteins.
- Proteins from both methods adopted folded conformations with anticipated secondary structures (3-α or 4β+α topologies).
- Recombinant and synthetic protein pairs exhibited similar conformations and highly comparable thermodynamic properties (Tm within 1 °C, ΔG < 0.1 kcal mol⁻¹).
Conclusions:
- Complementary recombinant and chemical synthesis methods provide reliable access to fold-switching proteins.
- These methods establish benchmarks for dual preparation strategies.
- Facilitates the study and design of fold-switching proteins with tunable properties for diverse applications.
