Work

HURDLER: a cloning method to overcome length limits in designed repeat proteins

Year: 2026

Type: article

Abstract: The number of internal repeat modules remains a major limiting factor in the construction and functional exploration of long repeat proteins. As a solution, we present High-Utility Recursive Directional Ligation for Extended Repeat proteins (HURDLER)-a modular cloning strategy combining codon optimization, seamless insertion of restriction sites, and modified recursive directional ligation. It presents a very robust technique to clone DNA for large repeat proteins, which may also contain non-repetitive parts, with high fidelity requiring a minimal number of individual cloning steps. Using HURDLER, we cloned designed armadillo repeat proteins with up to 85 internal modules, corresponding to >3700 residues. The method also generates intermediate variants with defined numbers of internal repeats, enabling systematic investigation of how the number of modules influences protein structure and function. HURDLER thereby provides a reliable solution to overcome restrictions in the overall number of internal modules used in the construction of very long repeat proteins. Such proteins are increasingly utilized as building blocks in macromolecular protein assemblies with defined structures.

Source: Nucleic Acids Research

DOI: 10.1093/nar/gkag988

Authors: Wenzhao Dai, Matthias Schuster, Oliver Zerbe

Institution: University of Zurich

Language: English

Topic: Biochemical and Structural Characterization

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,992

Funder: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung