Truncation-Free Genetic Code Expansion with Tetrazine Amino Acids for Quantitative Protein Ligations

Area of Science:

• Bioconjugation Chemistry
• Molecular Biology
• Chemical Biology

Background:

• Quantitative biomolecule labeling is crucial for antibody-drug conjugates, super-resolution microscopy, and protein complex stoichiometry.
• Bio-orthogonal labeling using genetically encoded noncanonical amino acids (ncAAs) is promising but limited by suboptimal reactivity and stability.
• Previous work introduced stable tetrazine (Tet)-containing ncAAs for rapid conjugation, but expression conditions affected protein reactivity.

Purpose of the Study:

• To address the limitations of incomplete conjugation caused by near-cognate suppression (NCS) and protein truncation.
• To develop improved machinery for robust and homogeneous production of reactive Tet-proteins.
• To demonstrate the utility of the developed system for rapid and homogeneous conjugation of biomedically relevant proteins.

Main Methods:

• Developed a more catalytically efficient aminoacyl tRNA synthetase (aaRS) and new machinery plasmids harboring the aaRS/tRNA pair.
• Utilized truncation-free cell lines and orthogonal synthetic origins for machinery vector compatibility.
• Investigated the impact of aaRS/tRNA pair plasmid copy-number on protein yield and quality.

Main Results:

• Identified reduction of on-protein Tet ncAAs and near-cognate suppression (NCS) as key factors limiting protein reactivity.
• Engineered new machinery plasmids enabling robust production of homogeneously reactive Tet-proteins, free from NCS and truncation.
• Demonstrated that aaRS/tRNA pair plasmid copy-number significantly influences protein production yields and quality.
• Successfully produced quantitatively reactive soluble Tet-Fabs, showcasing the system's application.

Conclusions:

• The developed aaRS/tRNA pair machinery plasmids overcome limitations in Tet-protein production, enabling homogeneous reactivity.
• This system facilitates rapid and homogeneous conjugation of biomedically relevant proteins, advancing applications in antibody-drug conjugation and imaging.
• The engineered system provides a versatile and compatible platform for quantitative biomolecule labeling.