Facilitated synthesis of proteins containing modified dipeptides

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Peptide and protein synthesis traditionally uses 20 standard amino acids.
• Incorporating non-proteinogenic amino acids (npAAs) expands protein functionality but faces challenges.
• Existing methods like chemical synthesis and ribosome-mediated synthesis have limitations.

Purpose of the Study:

• To develop novel strategies for improved yields of proteins containing npAAs.
• To overcome challenges in incorporating npAAs into contiguous positions within protein sequences.
• To enhance the utility of ribosome-mediated synthesis for creating modified proteins.

Main Methods:

• Selection of modified bacterial ribosomes for specific npAA recognition.
• Utilizing suppressor tRNAs (tRNA_CUA) activated with dipeptides of npAAs.
• Employing bis-aminoacylated suppressor tRNAs to enhance protein yields in vitro.
• Incorporating npAAs into contiguous positions within protein sequences.

Main Results:

• Demonstrated successful incorporation of npAAs into contiguous positions using modified ribosomes and dipeptide-activated suppressor tRNAs.
• Showcased the ability to synthesize proteins with npAAs not accessible via wild-type ribosomes.
• Extended the use of bis-aminoacylated tRNAs to npAAs, significantly improving protein yields.
• Successfully incorporated d-amino acids, β-amino acids, and dipeptidomimetic species.

Conclusions:

• Modified ribosomes and specialized tRNAs offer powerful tools for synthesizing proteins with diverse npAAs.
• These strategies overcome previous limitations in incorporating npAAs, particularly in contiguous arrangements.
• The developed methods significantly advance the field of protein engineering and synthetic biology.