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Abstract Using genetic code expansion (GCE) to encode bioorthogonal chemistry has emerged as a promising method for protein labeling, both in vitro and within cells. Here, we demonstrate that tetrazine amino acids incorporated into proteins are highly tunable and have extraordinary potential for fast and quantitative bioorthogonal ligations. We describe the synthesis and characterize reaction rates of 29 tetrazine amino acids (20 of which are new) and compare their encoding ability into proteins using evolved Tet ncAA encoding tRNA/RS pairs. For these systems, we characterized on-protein Tet stability, reaction rates, and ligation extents, as the utility of a bioorthogonal labeling group depends on its stability and reactivity when encoded into proteins. By integrating data on encoding efficiency, selectivity, on-protein stability, and in-cell labeling for Tet tRNA/RS pairs, we developed the smallest, fastest, and most stable Tet system to date. This was achieved by introducing fluorine substituents to Tet4, resulting in reaction rates at the 10⁶ M⁻¹s⁻¹ level while minimizing degradation. This study expands the toolbox of bioorthogonal reagents for Tet-sTCO-based, site-specific protein labeling and demonstrates that the Tet-ncAA is a uniquely tunable, highly reactive, and encodable bioorthogonal functional group. These findings provide a foundation to further explore Tet-ncAA encoding and reactivity.
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Tetrazine Amino Acid Encoding for Rapid and Complete Protein Bioconjugation
Generating protein conjugates using the bioorthogonal ligation between tetrazines and trans-cyclooctene groups avoids the need to manipulate cysteine amino acids, and the ligation is rapid, site-specific, stoichiometric and allows for labeling of proteins in complex biological environments. Here, we provide a protocol for the expression of conjugation-ready proteins at high yields in Escherichia coli with greater than 95% encoding and labeling fidelity. This protocol focuses on installing the “Tet2” tetrazine amino acid using an optimized genetic code expansion (GCE) machinery system, Tet2 “pAJE-E7”, to direct Tet2 encoding at TAG stop codons in BL21 E. coli strains, enabling reproducible expression of Tet2-proteins that quantitatively react with trans-cyclooctene (TCO) groups within 5 minutes at room temperature and physiological pH. Use of the BL21 derivative B95(DE3) minimizes premature truncation byproducts caused by incomplete suppression of TAG stop codons and this makes it possible to use more diverse protein construct designs. Here, using a superfolder green fluorescent protein construct as an example protein, we describe in detail a four-day process for encoding Tet2 with yields of ~200 mg per liter culture. Additionally, a simple and fast diagnostic gel electrophoretic mobility shift assay to confirm Tet2-Et encoding, and reactivity is described. Finally, strategies to adapt the protocol to alternative proteins of interest and optimize expression yields and reactivity for that protein are discussed.
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- Award ID(s):
- 2054824
- PAR ID:
- 10533065
- Publisher / Repository:
- Bio-protocols
- Date Published:
- Journal Name:
- BIO-PROTOCOL
- Volume:
- 14
- Issue:
- 1351
- ISSN:
- 2331-8325
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.21769/BioProtoc.5048
