Isopropylthio β d galactoside

Ultracompetent E. coli were transformed with pTrcHisB vectors coding the various PPX deletion mutants. Recombinant protein expression was induced by 0.5 mM isopropylthio-β-D-galactoside (Sigma-Aldrich) at 37 °C for 4 h. Bacteria were collected by centrifugation, resuspended in binding buffer (20 mM NaH₂PO₄, 500 mM NaCl and 20 mM imidazole, pH 7.4) and lysed by sonication. Cell lysates were centrifuged (10,000 g for 10 min at 4 °C) and supernatants were loaded on 1 ml HisTrap FF crude column (GE Healthcare). Following washing, bound proteins were eluted with 20 mM NaH₂PO₄, 500 mM NaCl and 500 mM imidazole, pH 7.4. Fractions containing mutants were combined, and solvent was changed to PBS, pH 7.4, using desalting columns (Econo-Pac 10 DG, Bio-Rad). Protein concentrations were determined by the Bradford method. Coomassie brilliant blue staining assessed protein purity. Western blotting was performed using 6xHis-tag antibody (1:1,000, Merck Millipore, catalogue number #70796-3) and horseradish peroxidase (HRP)-coupled anti-mouse Fc antibody (1:5,000, Jackson ImmunoResearch, #115-035-003). Full scans of the western blots are shown in Supplementary Fig. 4.

The expression and purification procedure of recombinant PPX_Δ12 was performed as described previously (21 (link)). In brief, DH5α competent E. coli cells were cultured at 37°C on an orbital shaker after transformation with plasmid DNA (pTrcHisB backbone) coding for PPX_Δ12. Induction of protein expression through the pTrc promoter was achieved by addition of 0.5 mM isopropylthio-β-D-galactoside (Sigma-Aldrich). After lysis of cells by sonication, protein purification was performed using an ÄKTA™ start FPLC system (GE Healthcare) connected to a 1 ml HisTrap FF crude column (GE Healthcare). Histidine-tagged proteins were eluted with a buffer containing 20 mM NaH₂PO₄, 500 mM NaCl and 500 mM imidazole. The buffer of the elution fractions was changed to PBS, pH 7.4.

EcoRI restriction sites were used to insert the PCR-amplified DNA fragment (obtained with primers described above) encoding for the PR domain N-terminal (PR-I), central (PR-II), and C-terminal (PR-III) domain fragments into pMAL-c5X vectors (New England Biolabs GmbH). Ultracompetent XL10-Gold E. coli were transformed with vectors coding for the maltose-binding protein (MBP)-fused constructs MBP-PR-I, MBP-PR-II, and MBP-PR-III or MBP only. Recombinant protein expression was induced by 0.5 mM isopropylthio-β-D-galactoside (Sigma-Aldrich) at 37 °C for 4 h^{61 (link)}. Bacteria were harvested by centrifugation, resuspended in column buffer (20 mM Tris HCl, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.4) supplemented with 0.5 mg/mL lysozyme and protease inhibitor cocktail (Abcam) and lysed by sonication. Cell lysates were centrifuged (20,000 × g for 20 min at 4 °C) and supernatants were loaded on 5 mL amylose resin column (New England Biolabs). Following washing, bound proteins were eluted with column buffer supplemented with 10 mM maltose. Fractions containing mutants were combined and protein concentrations were determined by the Bradford method. Coomassie brilliant blue staining assessed protein purity. Western blotting was performed using rabbit polyclonal anti-MBP antibody [1:1000,^{62 (link)}] and HRP-coupled donkey anti-rabbit antibody (1:5000, Jackson Immunoresearch, AB_2340585).