TP-Ring and ZN-Ring Systems Allow Efficient Protein Clamping, Solubilization and Stabilization

Improving recombinant protein stability remains a central challenge in biotechnology, as many proteins exhibit limited robustness during production and use. Here, we introduce two intramolecular "clamping" systems, termed ZN-ring and TP-ring, that exploit ultrastable noncovalent protein-protein interactions to constrain the N- and C-termini of a target protein. These systems are derived from the high-affinity complexes formed by ZNHIT3/NUFIP1 and TAH1/PIH1, respectively, whose interactions are driven by extensive hydrophobic interfaces. Proof-of-concept constructs demonstrate that intrachain clamping preserves native-like interactions and yields highly stable assemblies with melting temperatures exceeding 95 °C. When applied to a model protein, the TP-ring significantly enhances thermal stability, consistent with structural predictions suggesting additional stabilizing contacts. Furthermore, clamping markedly improves the soluble expression of a challenging polyethylene terephthalate hydrolase (PETase) in Escherichia coli, without compromising its catalytically active fold. These results establish protein clamping as a versatile strategy to enhance stability, solubility, and expression. Given their intrinsic robustness and broad compatibility with diverse targets, ZN- and TP-ring systems provide a promising platform for protein engineering, with potential applications in biotechnology and structural biology.