N-Cyclopropene-L-lysine

Oller-Salvia B, et al. Angew Chem Int Ed Engl, 2019, 158(32), 10844-10848.

Phage display is a powerful technique for evolving proteins and peptides with new functions. However, the chemical diversity of the molecules that can be evolved is often restricted by the limited diversity of the encoded ones.
The site-specificity of CypK can be integrated into phage display single-chain antibody variable fragments (ScFvs) via a pyrrolysine-tRNA synthetase (PylRS)/tRNA pair. In this method, CypK bound to the phage display protein is labeled through a rapid inverse electron demand Diels-Alder reaction with a tetrazine derivative.
The efficiency of displaying non-canonical amino acid (ncAA)-containing proteins is significantly improved by translating the displayed protein fusion from an orthologous ribosome binding site using an evolved ortholog. This optimized system allows for the binding of CypK to various sites in the ScFv in response to the amber codon and tetramer codon.
CypK is incorporated into different sites, allowing for the double labeling of ScFv with different probes in a one-pot assay through mutually orthogonal reactions. This enhancement expands the functional repertoire of phage display proteins and paves the way for broader applications of phage display technology.

Oller-Salvia B, et al. J Vis Exp, 2018, 139, 58066.

Cyclopropene-L-lysine (CypK) can be efficiently integrated into antibodies by genetic code expansion, and rapidly binds tetrazine derivatives by counter-requiring Diels-Alder cycloaddition to achieve site-specific, rapid and efficient attachment of tetrazine-containing molecules, thereby generating antibody-drug couplings.
The steps of antibody expression are as follows:
A vial of HEK suspension cells is thawed and transferred to a 250 mL flask containing 50 mL of expression medium supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 250 ng/mL amphotericin B. Cells are placed in a 37 ℃, 8% CO₂ incubator with a 125 rpm shaker. Before transfection, cells are passaged every 2-3 days to achieve a concentration of 0.3-0.5×10⁶ cells/mL and passaged at least 2 times.
When cell density reached 2.5×10⁶ cells/mL (2-3 days after splitting), a fresh 100 mM CypK solution is prepared: 64 mg of CypK is dissolved in 2.5 mL of 0.1 M NaOH, vortex-mixed, spun, and sonicated.
2.5 mL of 100 mM CypK (in 0.1 M NaOH) is added to 42.5 mL of expression medium with antibiotics, mixed, and then 250 µL of 0.1 M HCl is added and sterilized through a 0.22 µm filter.
50 µg of each HC and LC pKym1 plasmid are diluted to 2.5 mL with reduced serum medium. In another tube, 135 µL of transfection reagent is diluted to 2.5 mL with reduced serum medium. After 5 minutes, the plasmid and transfection reagent solutions are mixed and incubated for 20 minutes to form the DNA-transfection reagent complex.
Meanwhile, 125 million cells are centrifuged at 500 x g for 5 minutes and resuspended in expression medium with CypK. The DNA-transfection reagent mixture is added. After 20 hours of incubation, 250 µL of transfection reagent enhancer is added.
Antibodies are harvested from the supernatant 6-7 days after CypK addition (no media change required during expression).