Superdex 200 10 300

Analytical size-exclusion chromatography (SEC) of the full-length and trypsin-processed Vpb4Da2 was performed on an AKTA pure™ using a Superdex-200 10/300 GL increase (Cytiva) equilibrated at 4°C with 25 mM Na carbonate pH 9.5 and 100 mM NaCl. Protein samples were injected at the flow rate of 0.5 mL/min. Superdex-200 10/300 was calibrated using Ferritin (440, 000 Da), Aldolase (158, 000 Da), Conalbumin (75,000 Da), Ovalbumin (44, 000 Da), and Blue dextran 2000, which were all from Cytiva. Kav = (Ve-Vo)/Vc-Vo), with Ve = elution volume, Vo = void volume (8.07 mL), and Vc = Column volume (24 mL) were used. Calibration curve equation, Y = -03896x + 1.2061 (R² = 0.9225) was used to calculate the molecular weight for full length and trypsin processed Vpb4Da2.
Thermal stability of the purified wild-type Vpb4Da2 and variants in protein storage buffer was performed in triplicate reactions using 25 μL of 0.5 mg/mL protein and 5 μL of 10x SYPRO™ Orange (Thermo Scientific). The CFX96™ real-time PCR detection system (Bio-Rad) was used to follow a thermal denaturation gradient of 20°C to 95°C with an increment of 0.5°C per 5 Sec. Fluorescence data were fit using the CFX manager 2.1 (Bio-Rad).

The synthetic gene prkn2_rat (GeneArt) was cloned into pMK (kanR) using SacI and KpnI cloning sites. The full-length gene was cleaved by BamHI and XhoI restriction sites and ligated into pGEX-6P1 vector. The recombinant proteins of construct were overexpressed in E. coli KRX cells and grown at 37 °C in lysogeny broth medium containing 100 mg/l ampicillin until the A₆₀₀ level of 0.6 was recorded. Proteins expressions were induced by addition of 0.7 mM IPTG, supplemented with 0.5 mM ZnCl₂. Following induction, cells were grown overnight at 16 °C. The cells were harvested and lysed by sonication on ice in buffer A (20 mM Tris-HCl (pH 7.4), 120 mM NaCl) supplemented with 1% Triton X-100 and 2 mM PMSF.
The cell lysates were centrifuged at 48,000 g for 30 min, at 4 °C. The supernatant of GST-parkin was purified on a 20 ml GST column and eluted with 10 mM reduced glutathione. The pull-down fractions were concentrated using Amicon Ultra concentrators with 10 kDa Millipore membrane and separated by gel-filtration on Superdex200 10/300 (Cytiva, Uppsala, Sweden). Purified on affinity chromatography, parkin was eluted from size-exclusion Superdex200 column in V₀ as oligomers with molecular weight not less than 1000 kDa.