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Directed evolution of multimeric proteins is enabled by dual-compensatory gene duplication.

Rezwan Siddiquee1,2,3, Felicia Lie1,3, Taylor N Szyszka1,2,3

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Gene duplication enhances protein engineering by enabling selection of novel variants. This strategy overcomes limitations in current methods, expanding access to diverse, high-performing multimeric proteins.

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Area of Science:

  • Biochemistry
  • Evolutionary Biology
  • Protein Engineering

Background:

  • Gene duplication is a key evolutionary mechanism for protein diversification.
  • Current protein engineering methods do not leverage gene duplication for directed evolution.
  • This limits the discovery of novel multimeric protein variants.

Purpose of the Study:

  • To develop a novel directed evolution strategy for multimeric proteins using gene duplication.
  • To overcome metabolic burden and self-assembly fitness limitations in protein engineering.
  • To expand access to diverse, high-performing protein variants.

Main Methods:

  • Implemented a gene duplication strategy within a directed evolution framework.
  • Applied the method to a homomeric 240-mer capsid system.
  • Selected for extreme homomeric variants and obligate heteromers.

Main Results:

  • Successfully enriched previously inaccessible protein variants.
  • Demonstrated selection of both extreme homomeric variants and obligate heteromers.
  • Validated the strategy's effectiveness in expanding engineering access.

Conclusions:

  • Gene duplication is a powerful tool for protein engineering of multimeric proteins.
  • This strategy significantly broadens the scope of accessible protein variants.
  • The approach provides a model for natural evolutionary diversification of complex protein assemblies.