Mutagenesis and evolution of sulfated antibodies using an expanded genetic code

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Posttranslational modifications (PTMs) are crucial for protein function but challenging to study.
• Tyrosine sulfation is a key PTM affecting protein-protein interactions.
• Studying specific PTMs like sulfation requires specialized techniques.

Purpose of the Study:

• To develop a method for genetically encoding posttranslationally modified amino acids in E. coli.
• To characterize the role of tyrosine sulfation in the binding affinity of the anti-gp120 antibody 412d.
• To engineer novel antibodies with enhanced binding properties through directed evolution.

Main Methods:

• Genetically encoding unnatural amino acids using orthogonal aminoacyl-tRNA synthetase/tRNA pairs.
• Expressing selectively sulfated antibody variants in E. coli.
• Site-specific mutagenesis to assess the impact of individual tyrosine sulfates.
• Directed evolution to generate novel antibody variants.

Main Results:

• Demonstrated successful genetic encoding of sulfotyrosine in E. coli.
• Quantified the contribution of individual and dual tyrosine sulfates to 412d binding affinity for gp120 (4.5-fold, 212-fold, and 500-fold increases, respectively).
• Evolved novel doubly sulfated antibodies with subnanomolar affinity for gp120.

Conclusions:

• The developed strategy enables efficient biochemical study of PTMs.
• Tyrosine sulfation is critical for high-affinity binding of 412d to gp120.
• Genetically encoded unnatural amino acids are powerful tools for protein engineering and functional studies.