Xtractor buffer

N-terminal His6-tagged full-length human TAZ, full-length human NANOG, human NANOG-CAD and human NANOG-NAD plasmids were synthesized by chemical synthesis method and inserted into the NdeI/XhoI sites of pET-28a (+) plasmids and confirmed by DNA sequencing analysis. E. coli (DE3) were grown at 37 °C and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16 h at 20 °C. Cells were pelleted, resuspended, and lysed in the xTractor buffer (Cat#635623; Takara) supplemented with an EDTA-free protease inhibitor cocktail (Cat#87786, Thermo) by rocking at room temperature for 20 min. After centrifugation at 16,000 g for 30 min at 4 °C, the supernatant was collected. The soluble protein was purified using pre-balanced nickel columns (Cat#635623; Takara) and eluted with the following buffer: 20 mM Na₃PO₄, 500 mM NaCl, 500 mM imidazole, and pH 7.6. Amicon Ultra-0.5 spin columns (Cat#Z677094; Merck-Millipore) were used for further concentration and buffer exchange. Purified protein was quantified using a ND-2000C NanoDrop spectrophotometer (NanoDrop Technologies) with OD 280 nm and verified by Coomassie staining. Recombinant protein was diluted in storage buffer (50 mM Tris-HCl, 500 mM KCl, 1 mM DTT, and 5% glycerol), flash-frozen in liquid nitrogen, and stored at -80 °C.

PR-1-like protein derived from O. javanica (OPRP) was sub-cloned into a high yield expression vector system. The full-length OPRP gene sequence was cloned into Escherichia coli DH5α and successfully ligated into the expression vector pET32a in BL21 (DE3). BL21 cells harboring pET32a-OPRP were cultured in the presence of 10 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 6 h. Cell pellets were then collected from cultured cells and lysed with X-tractor buffer (Takara, Japan). Finally, OPRP was isolated using nickel-functionalized membranes that provide specific and highly sensitive detection of His-tagged fusion proteins (CapturemTM His-Tagged Purification Kit, Takara, Japan), followed by elution with endotoxin-free buffer containing imidazole and subsequently dialyzed to eliminate unwanted remaining molecules overnight at 4 °C. Purified proteins were confirmed with an electrophoresis system using 10% SDS-polyacrylamide gels and stained with 0.01% Coomassie Brilliant Blue R-250 (Sigma-Aldrich, St. Louis, MO, USA). OPRP was quantitated using a Bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) for further examination.

Ubiquitin-binding assay was performed essentially as described in [40 (link)]. Briefly, recombinant proteins were induced in E. coli with 1 mM IPTG at 37C for 4 hours. Cells were harvested and suspended in 1x PBS (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, and 1.8mM KH2PO4), followed by lysis in xTractor buffer (Takara) per the manufacturer’s protocol. Lysates were cleared by centrifugation and immobilized on TALON metal affinity resin (Takara) per the manufacturer’s protocol. Immobilized proteins were washed with 1x PBS and incubated overnight at 4C with clarified lysates containing various GST-fusion constructs. Bound proteins were washed four times with 1x PBS with 5mM imidazole and eluted by heating to 99C for 5 min in 1x SDS loading buffer. Eluted protein was run on a 4–20% Mini-PROTEAN(R) TGX Stain-Free Protein gel (Bio-Rad) and stained with Coomassie brilliant blue.

All manipulations were performed at 4 °C. Cells were harvested after cultivation by centrifugation at 13,200 rpm for 5 min and washed twice with sterile 0.9% NaCl.
Crude extracts were prepared using xTractor™ Buffer (Takara Bio, Mountain View, CA, USA) according to the manufacturer’s instructions.
The supernatants were decanted and then subjected to 12% SDS-PAGE. Protein molecular mass evaluation was performed by comparing with PageRuler Prestained Protein Ladder 26616 (Thermo Scientific, Waltham, MA, USA). Pure hexahistidine-tagged DSDs were isolated using the Capturem™ His-Tagged Purification Miniprep Kit (Takara Bio, Mountain View, CA, USA).

Overnight culture of the ΔczcR mutants with complementation of His₆-tagged czcR or czcR variants (D8A and D51A) was 1:100 diluted into 5-mL fresh LB medium and subcultured with or without the supplementation of 0.5 mM ZnSO₄ until OD₆₀₀ was 1.0. Approximately 2.0 × 10⁹ cells were harvested and lysed by 40 μL xTractor Buffer (TaKaRa) supplemented with protease inhibitor. After centrifugation, supernatant was mixed with SDS-loading buffer. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes by electroblotting. Membranes were blocked with Blocking buffer (5% nonfat milk PBST) followed by immunoblotting using anti-His₆ antibody (Abcam) and horseradish peroxidase-conjugated goat anti-rabbit antibody (TransGen Biotech). Proteins were detected using the ECL kit (Bio-Rad) according to the manufacturer’s protocol.

The expression plasmid pE-SUMO-vdUTPase, pE-SUMO-vdUTPaseD97A, and pE-SUMO-vUNG were used to transform E. coli Rosetta (Novagen). Plasmid-containing bacterial cultures were grown at 28 °C with shaking in Luria-Bertani (LB) medium containing 100 μg/mL of ampicillin. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added into the culture to a final concentration of 0.1 mM when the culture reached an absorbance of ~0.3 at 600 nm. The culture was incubated at 16 °C overnight and then harvested by centrifugation at 9250×g for 5 min at 4 °C, washed with PBS, and stored at −80 °C. Cell lysates were prepared by re-suspending the pellets in xTractor Buffer (Takara) and by sonicating briefly on ice. They were then clarified by centrifugation at 88,600×g for 20 min at 4 °C and the supernatant filtered through a 0.45-µm filter (Sartorius) prior to loading onto a Capturem™ His-Tagged Purification Miniprep Kit (Takara). After unbound proteins were washed away with two washes of wash buffer (20 mM Na₃PO₄, 150 mM NaCl, and pH 7.6) and once with wash buffer containing 40 mM imidazole, the target protein was eluted with elution-buffer A (20 mM Na₃PO₄, 500 mM NaCl, 500 mM imidazole, and pH 7.6). The eluted fraction was dialyzed twice with a desalting column (Apro Science) according to the manufacturer’s instructions and stored at −80 °C.