HURDLER: a cloning method to overcome length limits in designed repeat proteins
- 1Department of Chemistry, University of Zurich , Winterthurerstrasse 190, 8057 Zurich ,
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.
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BibTeX
@article{dai2026hurdler,
title = {{HURDLER: a cloning method to overcome length limits in designed repeat proteins}},
author = {Wenzhao Dai and Matthias Schuster and Oliver Zerbe},
journal = {Nucleic Acids Research},
year = {2026},
month = oct,
doi = {10.1093/nar/gkag988},
url = {https://academic.oup.com/nar/article/doi/10.1093/nar/gkag988/8915704},
note = {CC BY-NC 4.0},
}
RIS
TY - JOUR
TI - HURDLER: a cloning method to overcome length limits in designed repeat proteins
AU - Wenzhao Dai
AU - Matthias Schuster
AU - Oliver Zerbe
PY - 2026
DA - 2026/10/14
JO - Nucleic Acids Research
DO - 10.1093/nar/gkag988
UR - https://academic.oup.com/nar/article/doi/10.1093/nar/gkag988/8915704
AB - 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.
SN - 0305-1048
SN - 1362-4962
ER -