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. 2023 Nov 2;14:7001. doi: 10.1038/s41467-023-42810-5

Fig. 2. Synthetic, soluble, ditopic CC ligands result in robust receptor activation.

Fig. 2

a Schematic overview of the envisioned ligand-receptor interaction. N-termini linkage of two monomeric CCs results in the desired parallel conformation of a bivalent CC ligand for receptor activation. Arrow indicates N- to C- terminus directionality. b Schematic representation of the expression strategy of the monomeric CC ligand in E. coli. An N-terminus, cleavable small ubiquitin-like modifier (SUMO) tag is fused to a CC ligand, having a single cysteine at the C-terminus. For purification purposes, the fusion protein harbors an N-terminal hexahistidine (His) tag and a C-terminal Strep-tag, connected through a small, flexible glycine–serine (GS) linker (see “Methods” and Supplementary Fig. S4). c SDS-PAGE analysis showing the successful engineering of the dipeptide prior to purification (CCdimer(unp.). Anion-exchange chromatography results in removal of unreacted CC monomers (CCdimer(wash) and recovery of the ditopic CC ligands (CCdimer(pur.) (see “Methods” and Supplementary Fig. S4). d SEAP activity [U/L] in HEK293T cells transiently transfected with CC-GEMS receptors (A-typeJAK/STAT or B-typeJAK/STAT) with varied linker lengths αx of zero, four, eight, and 27 aa (GS or GSS repeats). Following transfection, cells were incubated with either 0.12 μM purified A’-A’ dipeptide or 0.24 μM A’ monomeric CC and SEAP expression was monitored to assess receptor activation (see “Methods”). Fold change and significance (two-tailed, unpaired t test) is noted above bars. ns: P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (see Supplementary Table S6). e Titration of ditopic ligand A’-A’ on HEK293T cells transiently transfected to express the A-typeJAK/STAT receptor with α2 linker (8 aa, GS repeats). Cells were incubated with a range of concentrations of ligand A’-A’ (0–1 μM) or 2 μM of monomeric A’ for 48 h. SEAP activity was monitored to assess receptor activation. Bars indicate mean activity; individual data points represent independent triplicates, performed on the same day. Source data are provided as a Source Data file.