Fig. 6: Purification and characterization of hTTR(59–127). | Communications Biology

Fig. 6: Purification and characterization of hTTR(59–127).

From: A serine protease secreted from Bacillus subtilis cleaves human plasma transthyretin to generate an amyloidogenic fragment

Fig. 6

a RP-HPLC purity check of hTTR(59–127). After lyophilisation, an aliquot of purified fragment was dissolved in 10 mM phosphate buffer, pH 7.4 and immediately injected onto an analytical C18 column, eluted (0.8 ml/min) with an acetonitrile-0.1%TFA gradient from 10 to 45% in 30 min. (Inset) SDS-PAGE (4–20% acrylamide) analysis of purified hTTR(59–127) (5 μg) under reducing conditions and Coomassie staining. For comparison, purified intact hTTR was also analysed. b Far-UV circular dichroism. Spectra of purified full-length hTTR and hTTR(59–127) fragment were recorded at a protein concentration of 0.1 mg/ml in 10 mM phosphate buffer, pH 7.4, at 25 °C. c DLS analysis. DLS measurements were carried out at a fragment concentration of 0.23 mg/ml. The arrow indicates the size of intact hTTR tetramer. DLS data are expressed as % intensity size distribution, while the values of Rh, %mass distribution and %PD are reported in the Supplementary Table 1. All measurements were carried out at 25 °C, immediately after dissolving hTTR(59–127) protein lyophilisate with phosphate buffer. Inset Native PAGE (5–12% acrylamide) of hTTR and hTTR(59–127) fragment (5 μg). For comparison, BSA was also loaded. d HDX-MS kinetics of deuterium uptake by intact hTTR (left panel) and hTTR(59–127) fragment (right panel). Proteins were incubated at 25 °C with 90% D2O in PBS buffer, pH 7.4, and m/z spectra were taken at increasing labeling time. For intact hTTR, the multiple charged species (z = +16) of S-Cys-hTTR (MW = 13,880.8 Da) was considered, whereas for hTTR(59–127) the deuterium uptake of the z = +8 species was monitored. For hTTR at t = 0, the species with m/z = 872.05 corresponds to the S-Cys-Gly-hTTR isoform (i.e., species 2:i in the Supplementary Fig. 1). A detailed description of the experimental procedure and data analysis is reported in the Supplementary Information. For clarity, the intensity of m0 and m100 spectra was downscaled by 4-fold, compared to the intensity of mt spectra.

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