Fig. 1: Protein purification and activation.
From: Molecular and in vivo studies of a glutamate-class prolyl-endopeptidase for coeliac disease therapy
a Purification of wild-type (WT) pro-neprosin by His6- or (b) Strep-tag affinity chromatography. The flow-through (FT), wash (W) and elution (E1–E3) fractions were analysed by SDS-PAGE and Coomassie staining, alongside molecular mass markers (lane M). Panels are representative of three independent experiments. c Pro-neprosin mutants (K118A, H134A, Y136A, Q173A, W175A, E188A, E188Q, Y214A, E297Q and E297A) after His6-tag affinity purification compared with the WT forms of (a) and (b). Figure representative of two independent experiments. d Size exclusion chromatography profiles of pro-neprosin with His6 tag (magenta), neprosin with Strep tag (green) and neprosin with His6 tag (blue) separated on a Superdex 75 10/300 GL column. Each curve is labelled with the elution volume in mL, representing monomers in all cases. e Autolytic maturation of pro-neprosin over time at 37 °C in an acidic buffer. Figure representative of two independent experiments. f Activation of pro-neprosin variants (Z lanes) by acidic autolysis (A lanes) or in trans by adding Strep-tagged neprosin (S lanes). Mutant K118A (third panel) was obtained as a pre-activated protein after affinity purification, revealing separate PD and mature protein bands (lane Z), which became fully activated by incubation in an acidic buffer (lane A). Figure representative of two independent experiments. For the distinct panels of this figure, relevant source data are provided as a Source Data file if adequate.
