Isopropyl β d thiogalactopyranoside

The Cas12m2 protein for structural analysis was expressed and purified using the protocol reported previously^{14 (link),20 (link)}. Briefly, the N-terminally His₆-tagged Cas12m2 protein was expressed in E. coli Rosetta 2 (DE3). E. coli cells were cultured at 37 °C until the OD₆₀₀ reached 0.8, and protein expression was then induced by the addition of 0.1 mM isopropyl β-d-thiogalactopyranoside (Nacalai Tesque). E. coli cells were further cultured at 20 °C overnight and collected by centrifugation. The cells were then resuspended in buffer A (20 mM HEPES–NaOH, pH 7.6, 20 mM imidazole and 1 M NaCl), lysed by sonication and centrifuged. The supernatant was mixed with 3 ml Ni-NTA Superflow resin (Qiagen), and the mixture was loaded into an Econo-Column (Bio-Rad). The protein was eluted with buffer B (20 mM HEPES–NaOH, pH 7.6, 0.3 M imidazole, 0.3 M NaCl) and then loaded onto a 5-ml HiTrap SP HP column (GE Healthcare) equilibrated with buffer C (20 mM HEPES–NaOH, pH 7.6, and 0.3 M NaCl). The protein was eluted with a linear gradient of 0.3–2 M NaCl and further purified by chromatography on a Superdex 200 column (GE Healthcare) equilibrated in buffer D (20 mM HEPES–NaOH, pH 7.6, 0.5 M NaCl). The purified proteins were stored at −80 °C until use. The crRNA was transcribed in vitro with T7 RNA polymerase and purified by 10% denaturing (7 M urea) polyacrylamide gel electrophoresis.

The PspA gene was amplified by polymerase chain reaction (PCR) and cloned into pET16b plasmid (Novagen, Darmstadt, Germany), as previously described, to yield pET16b-PspA plasmid [17 (link)]. To obtain PspA recombinant proteins, the plasmids were transformed into E. coli strain BL21 (DE3) (Novagen). Protein production was induced by adding isopropyl-β-D-thiogalactopyranoside (Nacalai Tesque, Kyoto, Japan). The culture pellets were sonicated for 1 min three times in buffer A (10 mM Tris-HCl [pH 8.0], 400 mM NaCl, 5 mM MgCl₂, 0.1 mM PMSF, 1 mM 2-mercaptethanol, and 10% glycerol). After centrifugation of the mixture at 4 °C and 17,800× g for 15 min, the supernatants were filtered through a 0.45 µm Millex-HV filter unit (Merck Millipore, Burlington, MA, USA) and loaded into HiTrap HP columns (GE Healthcare, Pittsburgh, PA, USA). PspA was eluted with buffer A containing 100 to 500 mM imidazole. The eluted protein was loaded into a PD-10 column (GE Healthcare) for exchange with PBS (Nacalai Tesque). The concentration of purified protein was measured by using a BCA protein assay kit (Pierce Chemical, Rockford, IL, USA). The purity of the eluted protein was confirmed in a NuPAGE electrophoresis system (Life Technologies, Carlsbad, CA, USA) followed by staining with Coomassie brilliant blue (Nacalai Tesque).

The entire coding sequence of human wildtype PTEN was amplified from a human cDNA library by the polymerase chain reaction (PCR). The cDNA encoding PTEN was subcloned into the pGEX-6P-1 vector. The recombinant human PTEN was expressed as an N-terminal glutathione S-transferase (GST)-tagged fusion protein in BL21 (DE3) cells transformed with the pGEX-PTEN vector. Protein production was induced by 0.5 mM isopropyl β-D-thiogalactopyranoside (Nacalai Tesque) at 25 °C for 12 h. GST-PTEN was affinity purified on Glutathione 4B Sepharose, eluted with 10 mM reduced glutathione in 50 mM Tris-HCl (pH 7.5), 150 mM sodium chloride and 1 mM dithiothreitol (DTT). Thiol groups oxidized during purification were reduced by incubation with 20 mM DTT for 1 h. Free glutathione and DTT were removed by buffer exchange to 50 mM potassium phosphate buffer (pH 7.0) using an ultrafiltrator (Piece Concentrators 9 K; Thermo Fisher Scientific). Samples were stored in at −80 °C before use.

GST-fusion proteins constructed in pGEX-6P-1 (GE Healthcare Life Sciences) were transformed into Escherichia coli BL21 (DE3) cells, which were then cultured at 37 °C in Luria–Bertani medium supplemented with 100 μg mL⁻¹ ampicillin and induced for 3 h at 37 °C with 0.3 mM isopropyl β-d-thiogalactopyranoside (Nacalai Tesque). Cells were harvested by centrifugation, washed with PBS, lysed in 40 mM Tris-HCl (pH 7.5), 5 mM EDTA, and 0.5 % Triton X-100, sonicated, and cleared by centrifugation. The resulting supernatant was incubated with Glutathione Sepharose 4 Fast Flow beads (GE Healthcare Life Sciences) for 2 h at 4 °C. After several washes with buffer, the beads were used directly in pulldown assays.

Recombinant His-tagged IcaR and IcaR_Jm were purified as described by Jeng et al. (6 (link)) with a few modifications. The icaR gene was amplified by PCR from the genomic DNA of FK300 or JP080 using primers pET-22b-IcaR-F and pET-22b-IcaR-R (Table 2) and cloned into the expression vector pET-22b(+) (Novagen). E. coli BL21(DE3) cells, transformed with the recombinant plasmid, were grown in 300 mL LB medium containing 100 μg/mL ampicillin at 37°C to an optical density of 0.5 at 600 nm. Protein expression was induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (Nacalai Tesque, Inc., Kyoto, Japan), and cells were harvested after incubation at 30°C for 2 days. Subsequently, the harvested cells were lysed by sonication on ice. His₆-tagged IcaR and IcaR_Jm were purified from the supernatant using Talon metal affinity resins (Clontech Laboratories, Inc.) according to the manufacturer’s protocol. Protein expression and purity were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 12% gel. Protein concentrations were measured using the Bio-Rad protein assay (Bio-Rad, Hercules, CA), with bovine serum albumin as the standard.