Thrombin enzyme

These scaffold proteins were expressed with a thrombin-cleavable N-terminal His₆ tag in BL21 (DE3) E. coli induced with 0.5 mM isopropyl -D-1-thiogalactopyranoside at OD₆₀₀ of 0.6 at 16 °C for 16 h. Cells were harvested, pelleted and stored at −20 °C. Cells were lysed using an Avestin EmulsiFlex C3 cell crusher and centrifuged at 4 °C, 13,000g for 40 min to remove insoluble cell debris. Scaffold proteins were purified from the soluble fraction by affinity chromatography over Talon Superflow resin (Takara Clontech), which was equilibrated with 20 mM Tris-HCl, 150 mM NaCl, and 5 mM imidazole at pH 7.4 and were eluted with 400 mM imidazole in 20 mM Tris-HCl, 150 mM NaCl at pH 7.4. The His₆ fusion partner was cleaved by thrombin enzyme (Sigma-Aldrich). Scaffold proteins were further purified using a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare), where it elutes as a single monomeric peak in 20 mM Tris-HCl, 150 mM NaCl at pH 7.4. Collected fractions were pooled, buffer exchanged to 50 mM Imidazole, 10 mM CaCl₂ and pH 6.8 for recording NMR experiments.

Plasmids used in the SPR assays were pGEX-4T2 cloned with DUSP3 cDNA (WT or the C124S mutant) (Torres et al., 2017 (link)), NPM full length (donated by Prof. Mitsuru Okuwaki, University of Tsukuba, Tsukuba-Ibaraki, Japan), ERK1 (donated by Prof. Rony Seger, The Weizmann Institute of Science, Israel), and pET21a(+) cloned with cDNA of truncated N-terminal of NPM_9–122 (donated by Se Won Suh, Addgene plasmid #23142). For dual-luciferase reporter assays, we used pGL3-p53RE (donated by Prof. Tomas Mustelin, Sanford Burnham Prebys Institute, La Jolla-CA, United States) and pRL-SV40 vectors (donated by Prof. Carlos F. M. Menck, Institute of Biomedical Sciences—University of São Paulo, São Paulo-SP, Brazil). Recombinant proteins were expressed in BL-21 (DE3) bacteria induced by 1 mmol/L IPTG for 3 h. DUSP3 (WT, C124S), and NPM full length proteins GST-tagged were purified by affinity chromatography in Glutathione-Sepharose 4B resin (GE Healthcare), and NPM_9–122 6x-His-tagged was purified on His-Trap^TM (Sigma-Aldrich, St. Louis-MO, United States), following the manufacturer protocol. The GST-tag was specifically removed by thrombin enzyme (Sigma-Aldrich, St. Louis-MO, United States) cleavage for 18 h at 18°C and purified by molecular weight exclusion filters (Millipore).

Human wt α-syn was expressed and purified as previously described [30 (link)–32 (link)] . E.coli BL21 bacteria (Invitrogen) was used for GST-α-syn fusion construct expression in the pGEX-4 T1 vector (kindly provided by Dr. Hyangshuk Rhim, the Catholic University College of Medicine, Seoul, Korea). The supernatant was kept for purification with affinity chromatography using glutathione sepharose beads (Amersham-Sweden). The GST-α-syn bound to beads was cleaved by thrombin enzyme (Sigma-Aldrich, USA). For thrombin removal of solution, benzamidine sepharose beads (Amersham-Sweden) were used. After centrifugation, the concentration of the resulted pure α-syn protein was purified by reverse phase HPLC, using analytical phenomenex jupiter C4 columns. Protein concentration was estimated by BCA assay (Pierce-USA) and the homogeneity and purity of α-syn was then estimated by analytical HPLC and SDS-PAGE.