Progress toward the evolution of an organism with an expanded genetic code

Area of Science:

• Molecular Biology
• Biochemistry
• Synthetic Biology

Background:

• Site-specific incorporation of unnatural amino acids into proteins is crucial for expanding protein functionality.
• Existing methods often face limitations in efficiency and scope within living systems.
• Development of orthogonal transfer RNA (tRNA) and aminoacyl-tRNA synthetase (aaRS) pairs is key for in vivo applications.

Purpose of the Study:

• To establish a general method for site-specific incorporation of unnatural amino acids into proteins in vivo.
• To develop a fully orthogonal tRNA/synthetase pair in Escherichia coli.
• To create a system for selecting mutant aaRS for charging novel molecules onto orthogonal tRNAs.

Main Methods:

• Derived an orthogonal suppressor tRNA from Saccharomyces cerevisiae tRNA2Gln.
• Established the orthogonality of the yeast tRNA/synthetase pair in E. coli.
• Developed a method to select mutant aaRS for unnatural amino acid charging.
• Created a nonradioactive screen for unnatural amino acid uptake.

Main Results:

• Successfully created an orthogonal yeast tRNA/GlnRS pair functional in E. coli.
• Demonstrated that the yeast tRNA is not charged by E. coli synthetases but is by yeast GlnRS.
• Developed a selection method for mutant aaRS capable of charging various molecules.
• Identified uptake of most glutamine and glutamic acid analogs by E. coli.

Conclusions:

• The developed orthogonal yeast tRNA/synthetase pair provides a robust system for in vivo unnatural amino acid incorporation.
• The methodology enables the expansion of the amino acid repertoire available for protein engineering.
• The findings facilitate the creation of novel proteins with tailored functions.