Extended Data Fig. 1: Characterization of engineered proteoglycan proteins.
(A) SDS-PAGE analysis and subsequent Coomassie (top) and fluorescence (bottom) imaging of SDC1, SDC2, SDC3 and SDC4 ectodomains reacted with TMR-azide confirms successful incorporation of pPY. As highly disordered proteins, SDCs run anomalously by SDS-PAGE. Representative of two technical replicates, molecular weight ladder in kDa. (B) Western blot analyses of SDC1 ectodomains probed with anti-SDC1 clone 281-2. (C) Intact mass spectrometry of SDC ectodomains confirms expected masses and demonstrates fidelity of pPY incorporation. Representative of two technical replicates, molecular weight ladder in kDa. N-terminal MQ residues were missing in some constructs, consistent with production and processing by E. coli. (D) Fluorescence microscopy images of beads following treatment with proteases or buffer with p-aminophenylmercuric acetate (PAPMA, required to activate MMP) shows SDC137TMR is only cleaved by MMP9 and trypsin (positive control). The TMR fluorophore is released from the beads after being washed, as it does not contain a poly-His tag sequence that enables the C-terminal fragment to remain anchored onto the nickel bead. (E) Circular dichroism (CD) spectrum of SDC1 ectodomains show >57% disordered conformations. APEX2 fusion proteins (A-SDC145,47 and A-SDC137,45,47) display enhanced helical structure relative to their non-fused counterparts due to the helical APEX220 domain. Ten readings were taken per sample to create an average CD spectrum. (F) Quantification of predicted secondary structures, generated by CAPITO analysis57.
