Immobilized Proteins

For purification of His₆-tagged CPD fusion proteins, overnight cultures of the appropriate strain were diluted 1∶500 into 1 L 2YT media and grown shaking at 37°C. When an OD₆₀₀ of 0.6 was reached, IPTG was added to 250 µM, and cultures were grown for 3-4 hrs at 30°C. Cultures were pelleted, resuspended in 25 mL lysis buffer [500 mM NaCl, 50 mM Tris-HCl, pH 7.5, 15 mM imidazole, 10% glycerol] and flash frozen in liquid nitrogen. Lysates were thawed, then lysed by sonication and cleared by centrifugation at 15,000×g for 30 minutes. His₆-tagged CPD fusion proteins were affinity purified by incubating the lysates in batch with 0.5–1.0 mL Ni-NTA Agarose beads (Qiagen) with shaking for 2–4 hrs at 4°C. The binding reaction was pelleted at 1,500×g, the supernatant was set aside, and the pelleted Ni²⁺-NTA agarose beads were washed three times with lysis buffer. In some cases, 10% of the Ni²⁺-NTA beads containing immobilized CPD-His₆ fusion proteins were removed, pelleted and then His₆-tagged fusion protein eluted using high imidazole buffer [500 mM NaCl, 50 mM Tris-HCl, pH 7.5, 175 mM imidazole, 10% glycerol].
To liberate untagged target proteins into the supernatant fractions, 300–500 µL lysis buffer was added to the Ni²⁺-NTA beads containing CPD-His₆ fusion proteins and the indicated amount of inositol hexakisphosphate (InsP₆, Calbiochem) was added. In general, on-bead cleavage was allowed to proceed by nutating the beads in the presence of 50–100 µM InsP₆ for 1–2 hr at either room temperature or 4°C. The beads were pelleted at 1,500×g, and the supernatant fraction was removed. The beads were then washed 3–4 times with 300–500 µL lysis buffer, and supernatant fractions retained. His₆-tagged proteins remaining on the beads (i.e. cleaved CPD-His₆) were eluted using high imidazole buffer [500 mM NaCl, 50 mM Tris-HCl, pH 7.5, 175 mM imidazole, 10% glycerol] in 300–500 µL volumes. The elution was repeated 3–4 times, and eluate fractions were collected. Purification of His₆-tagged proteins lacking the CPD was performed in parallel.
This general procedure was followed with the following exceptions: for purification of MMP12 constructs, the cultures were grown at 16°C for 8 hr after IPTG induction, and 1 mM tris(2-carboxyethyl)phosphine (TCEP) was added to the lysis buffer to prevent misfolding of the protein. PfSENP1 and BirA protein purifications were performed exclusively at room temperature, since at 4°C, protein aggregation was observed. For removal of the His₆-tag from His₆-PfSENP1, thrombin beads (Calbiochem) that had been washed in PBS were added to the eluted His₆-PfSENP1, which had been buffer exchanged into PBS according to the manufacturer's instructions. Thrombin cleavage was allowed to proceed with shaking overnight for 12 hr at room temperature. Aliquots were taken before and after thrombin addition to monitor cleavage efficiency. Thrombin cleaved, untagged PfSENP1 was enriched by performing a subtractive Ni²⁺-NTA pull-down. Untagged PfSENP1 from both methods was then buffer-exchanged into gel filtration buffer (50 mM NaCl, 20 mM Tris pH 8.0). Protein purifications were analyzed by SDS-PAGE and Coomassie staining using GelCode Blue (Pierce). Purified protein concentrations of purified were determined by Bradford assay (Pierce).

Proteins were resolved by reducing 15% SDS-PAGE and immunoblotted onto PVDF membranes. The immobilized Hh proteins were detected with a primary polyclonal anti-Hh antiserum (rabbit IgG, Santa Cruz Biotechnology, United States) and visualized with a secondary peroxidase-conjugated donkey anti-rabbit IgG (Dianova, Hamburg, Germany) followed by chemiluminescent detection. The signals were quantified with ImageJ software. Gel filtration analysis was carried out by FPLC (Äkta protein purifier, GE Healthcare, United States) on a Superdex200 10/300 GL column (Pharmacia) equilibrated with PBS at 4°C. Eluted fractions were TCA-precipitated, resolved by SDS-PAGE and immunoblotted. Signals were quantified using ImageJ and values expressed relative to the strongest signal, which was set to 100%.

Whole‐cell lysates were obtained from RASSF1 and control shRNA transfected HEK293FT cells treated with tunicamycin (5 μg·mL⁻¹) for 5 h. The cells were harvested with RIPA lysis buffer (Thermo Fisher, Waltham, MA, USA). Lysates were sonicated for 30 s, and the protein concentration was measured by DC protein assay (Bio‐Rad). Protein samples (30 μg each) were separated by 4–12% PAGE Gel (GenScript, Piscataway, NJ, USA) and then transferred onto PVDF membranes (Millipore‐Sigma, Burlington, MA, USA). Membranes were blocked with 5% nonfat milk for 60 min at room temperature and incubated overnight at 4 °C with recombinant anti‐RASSF1 rabbit monoclonal antibody (1 : 500, ab126764; Abcam) or anti‐α‐Tubulin monoclonal mouse antibody (1 : 5000, T9026; Sigma). After washing, membranes were incubated for 1 h at room temperature with horse radish peroxidase (HRP) conjugated goat anti‐rabbit IgG secondary antibody (1 : 10 000; Abcam) or goat anti‐mouse IgG secondary antibody (1 : 10 000; ThermoFisher) at room temperature. The immobilized proteins were detected using the enhanced chemiluminescence reagent plus (PerkinElmer, Waltham, MA, USA). Images were obtained with ChemiDoc™ Touch Imaging System (Bio‐Rad) and analyzed with image lab.

The purification of GST-tagged fusion proteins and in vitro translation of AurA were performed as described previously (28 (link)). Transformants harboring pGEXT2T:HPVE6s constructs were grown overnight. The culture was then inoculated into fresh medium and incubated for 1 h. Recombinant protein expression was then induced by addition of isopropyl-β-d-thiogalactopyranoside (IPTG, Sigma) to a final concentration of 1 mM and incubated for a further 3 h. The bacteria were harvested and lysed with cold 1× PBS containing 1% Triton X-100, and sonicated twice for 30 s. The supernatants were collected and incubated with glutathione-conjugated agarose resin on a rotating wheel overnight at 4°C. After extensive washing, the amount of immobilized GST fusion proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained by GelCode Blue Stain Reagent (Thermo Fisher Scientific).
AurA protein was translated in vitro using the TNT Coupled Reticulocyte Lysate System (Promega), according to the manufacturer’s recommendation. Equal amounts of in vitro-translated AurA were added to purified GST fusion proteins and incubated for 1 h at room temperature. After being washed 3 times with PBS containing 0.1% Tween 20, the bound proteins were subjected to SDS-PAGE and Western blotting for detection of AurA.