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An expanded genetic code for the characterization and directed evolution of tyrosine-sulfated proteins

Abstract

Tyrosine sulfation, a post-translational modification that enhances extracellular protein-protein binding, plays a crucial role in various bacterial and viral infections. However, studying tyrosine-sulfated proteins can be difficult, because tyrosine sulfation of proteins often leads to mixtures of different sulfation patterns. In addition, efforts to engineer tyrosine-sulfated proteins is limited, because sulfation of the correct tyrosine is coupled to its local amino acid sequence contexts, termed sulfation motifs, which cannot be mutated without changing the sulfation efficiency.

These complications can be resolved using an expanded genetic code system. A cell with an expanded genetic code for sulfotyrosine (sY) decouples sulfation motifs from the expression of homogenously sulfated proteins; this is accomplished through the site-specific incorporation of sY as a 21st amino acid at amber stop codons. Here, we use an upgrade expanded genetic code system to express high amounts of tyrosine-sulfated proteins for several applications. First, we expressed homogenously sulfated, bacterial RaxX peptide in order to validate its role in triggering immune response in rice plants. Second, we expressed all 32 possible sulfoforms of E51, one of the best sulfated anti-HIV antibodies at neutralizing HIV infections, and characterized their ability to bind to HIV's coat protein, gp120. We found several E51 sulfoforms that bind to gp120 with similar affinities as naturally sulfated E51 and determined key E51 residues for binding gp120. Lastly, we conducted multiple directed evolution experiments to improve the gp120-binding of E51 by repurposing E51's sulfation motifs with residues that bind to gp120.

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