In vitro genetic code reprogramming and expansion to study protein function and discover macrocyclic peptide ligands.

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• In vitro translation systems allow the incorporation of non-canonical amino acids (ncAAs) into proteins.
• Techniques such as sense codon reprogramming, stop codon suppression, and breaking codon degeneracy enable ncAAs incorporation.

Purpose of the Study:

• To review methods for introducing ncAAs into proteins using in vitro translation.
• To discuss the application of ncAAs in creating proteins with novel properties and studying protein function.
• To explore the tolerance of the translation apparatus to artificial building blocks.
• To highlight the combination of ncAAs with mRNA-displayed peptide libraries for ligand discovery.

Main Methods:

• Sense codon reprogramming
• Stop codon suppression
• Breaking codon degeneracy
• mRNA-displayed peptide libraries

Main Results:

• Proteins with novel properties can be created.
• Sophisticated studies of protein function are facilitated.
• The tolerance of the translation apparatus to artificial building blocks can be explored.
• Protease-stable, macrocyclic peptide libraries for ligand discovery can be generated.

Conclusions:

• In vitro translation systems are powerful tools for incorporating ncAAs.
• ncAAs enable the creation of proteins with enhanced properties and facilitate functional studies.
• Combining ncAAs with display technologies opens new avenues for drug discovery.