Fast analysis and engineering of protein function by microbe-independent deep assembly and screening

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Traditional protein engineering methods in mammalian cells are hindered by time-consuming and labor-intensive plasmid cloning procedures.
• Efficient sequence-fitness analysis of large protein variant libraries is crucial for optimizing protein function.

Purpose of the Study:

• To introduce Microbe-Independent Deep Assembly and Screening (MIDAS), a novel platform for rapid, plasmid-free protein variant expression and characterization in mammalian cells.
• To demonstrate the versatility of MIDAS for deep mutational scanning, combinatorial assembly, and protein engineering.

Main Methods:

• Developed MIDAS, a deterministic platform that bypasses microbial cloning by directly transfecting PCR-assembled genes into mammalian cells.
• Applied MIDAS for engineering an acetylcholine neurotransmitter bioluminescent indicator (ACh-NeuBI) and improved NanoLuc luciferase variants.
• Utilized MIDAS for sequence-fitness assessment of arbitrary mutational spaces, including deep saturation mutagenesis.

Main Results:

• MIDAS enables high-quality sequence-fitness assessment in less than one workday from PCR to cell transfection.
• Engineered a high-performance ACh-NeuBI with stepwise improvements in responsivity.
• Developed improved NanoLuc luciferase variants and characterized the structural basis of mutational tolerance and substrate specificity.

Conclusions:

• MIDAS is a versatile and rapid method for plasmid-free protein engineering and sequence-fitness analysis in mammalian cells.
• Offers a practical alternative to cloning-based approaches for protein optimization and characterization.
• Facilitates efficient exploration of mutational landscapes for protein design.