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; this ligation is rapid, site-specific, and 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 min at room temperature and physiological pH. The use of the BL21 derivative B95(DE3) minimizes premature truncation byproducts caused by incomplete suppression of TAG stop codons, which 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 of culture. Additionally, a simple and fast diagnostic gel electrophoretic mobility shift assay is described to confirm Tet2-Et encoding and reactivity. Finally, strategies are discussed to adapt the protocol to alternative proteins of interest and optimize expression yields and reactivity for that protein. Key features • Protocol describes site-specific encoding of the tetrazine amino acid Tet2-Et into proteins for bioorthogonal, quantitative, and rapid attachment of trans-cyclooctene-containing labels. • Protocol uses auto-induction methods for the production Tet2-Et protein in E. coli. • This protocol focuses on Tet-protein expressions in BL21(DE3) and B95(DE3) strains, which take approximately 4 days to complete. • SDS-PAGE mobility shift assay using a strained TCO-PEG5000 (sTCO-PEG5000) reagent provides a simple, generalizable method for testing Tet-protein reactivity.

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

Quantitative labeling of biomolecules is necessary to advance areas of antibody-drug conjugation, super-resolution microscopy imaging of molecules in live cells, and determination of the stoichiometry of protein complexes. Bio-orthogonal labeling to genetically encodable noncanonical amino acids (ncAAs) offers an elegant solution; however, their suboptimal reactivity and stability hinder the utility of this method. Previously, we showed that encoding stable 1,2,4,5-tetrazine (Tet)-containing ncAAs enables rapid, complete conjugation, yet some expression conditions greatly limited the quantitative reactivity of the Tet-protein. Here, we demonstrate that reduction of on-protein Tet ncAAs impacts their reactivity, while the leading cause of the unreactive protein is near-cognate suppression (NCS) of UAG codons by endogenous aminoacylated tRNAs. To overcome incomplete conjugation due to NCS, we developed a more catalytically efficient tRNA synthetase and developed a series of new machinery plasmids harboring the aminoacyl tRNA synthetase/tRNA pair (aaRS/tRNA pair). These plasmids enable robust production of homogeneously reactive Tet-protein in truncation-free cell lines, eliminating the contamination caused by NCS and protein truncation. Furthermore, these plasmid systems utilize orthogonal synthetic origins, which render these machinery vectors compatible with any common expression system. Through developing these new machinery plasmids, we established that the aaRS/tRNA pair plasmid copy-number greatly affects the yields and quality of the protein produced. We then produced quantitatively reactive soluble Tet-Fabs, demonstrating the utility of this system for rapid, homogeneous conjugations of biomedically relevant proteins.