Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Genetic code expansion allows incorporating unnatural amino acids into proteins for protein engineering.
• Cell-free systems offer advantages over in-vivo methods but are often complex to prepare and use.
• Current limitations hinder the widespread adoption of cell-free genetic code expansion.

Purpose of the Study:

• To develop a simplified method for preparing cell-free systems for genetic code expansion.
• To establish a modular approach for utilizing cell-free genetic code expansion systems.
• To demonstrate the advantages of cell-free systems for incorporating specific unnatural amino acids.

Main Methods:

• Developed a simplified protocol for preparing cell-free extracts from Escherichia coli.
• Implemented a modular system for preparing and storing translational components.
• Demonstrated simultaneous incorporation of two unnatural amino acids into a reporter protein.
• Showcased the incorporation of a specific unnatural amino acid into ubiquitin using a specialized tRNA/tRNA-synthetase pair.

Main Results:

• The simplified preparation method yields efficient cell-free extracts for genetic code expansion.
• The modular approach allows for long-term storage (over 4 years) and flexible combination of components.
• Successfully achieved simultaneous incorporation of two distinct unnatural amino acids.
• Incorporated δ-thio-boc-lysine into ubiquitin, a feat not possible in living cells.

Conclusions:

• This work presents a simplified and modular cell-free system for genetic code expansion.
• The developed system overcomes limitations of previous methods, enhancing accessibility and efficiency.
• Cell-free genetic code expansion provides unique capabilities not achievable in living systems, expanding protein engineering possibilities.