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

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.