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

HURDLER enables cloning of designed armadillo repeat proteins with up to 85 internal modules, exceeding previous length limits and facilitating structural studies.

• Why it matters: Long repeat proteins are valuable for building macromolecular assemblies, but their construction is limited by the difficulty of cloning extensive internal repeats, hindering functional exploration.
• What they did: The authors developed HURDLER, a modular cloning method combining codon optimization, seamless restriction site insertion, and recursive directional ligation, to efficiently assemble large repeat proteins.
• The result: Using HURDLER, they successfully cloned proteins with over 3700 residues and generated variants with specific repeat numbers, enabling systematic analysis of how repeat length affects structure and function.