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. 2024 Jul 12;4(4):190–203. doi: 10.1021/acsbiomedchemau.4c00026

Figure 2.

Figure 2

Production of functional dimeric and multimeric MK1-3.6.10 using recombinant monomeric MK1-3.6.10. (a) Construct design for recombinant head-to-tail dimeric MK1-3.6.10. In the di-MK1-3.6.10GS construct, two DCPs were separated by a GSGSGS linker and TEV protease digestion would produce one major DCP species with six disulfide bonds. For the control construct, di-MK1-3.6.10TEV, two DCPs were separated by the TEV protease digestion site and TEV protease digestion would produce two monomeric DCPs (MK1-3.6.10-SENLYFQ and MK1-3.6.10) with three disulfide bonds in each if the two DCPs fold independently without forming any intermolecular disulfide bonds. (b) LC–MS and SDS PAGE analysis of di-MK1-3.6.10GS and di-MK1-3.6.10TEV expression. di-MK1-3.6.10GS produced one major species with six disulfide bonds, with a minor species (∼10%) also containing six disulfide bonds. di-MK1-3.6.10TEV produced two DCPs with three disulfide bonds in each. (c) Different forms of recombinant dimeric MK1-3.6.10 (head-to-tail) show similar affinity to their synthetic counterparts. The affinity values were measured by SPR. All values were averaged from three independent runs. (d) Recombinant head-to-tail MK1-3.6.10 dimers do not activate Wnt signaling in the TOPbrite reporter cells. Representative data from three independent runs are shown. (e) Generalized reaction scheme for STL. (f) Different forms of head-to-head dimeric MK1-3.6.10, produced through STL, show similar affinity to their synthetic counterparts. The affinity values were measured by SPR. All values were averaged from three independent runs. (g) Head-to-head dimeric and multimeric MK1-3.6.10 can activate Wnt signaling in the reporter assay. Representative data from three independent runs are shown.