Gst tag protein purification kit

Competent E. coli BL21‐CodonPlus (DE3)‐RIPL strain were transformed with pEN‐GST‐SRSF5‐flag, pEN‐GST‐U1A‐His, pEN‐GST‐U1–70k‐His, pEN‐GST‐U1C‐His and empty vectors. Single colony of each construct was then grown in LB media at 37 °C until desired density, and then induced with 0.3 mm IPTG (Beyotime Biotechnology, #ST098) at 30 °C overnight. GST‐tagged proteins were purified using standard protocols with the GST‐tag Protein Purification Kit (Beyotime Biotechnology, #P2262) according to the manufacturer's instructions. For GST‐SRSF5‐flag, the GST‐tag was removed by 20 units of PreScission Protease (2 U µL⁻¹; Beyotime Biotechnology, #P2302) at 4 °C overnight in 50 mm Tris‐HCl, 150 mm NaCl, 1 mm EDTA, 1 mm DTT, pH7.5. For GST pulldown, 300 ng of purified recombinant SRSF5‐Flag was incubated with 150 ng of glutathione bound GST‐tagged U1A‐His, U1‐10K‐His, U1‐C‐His or with negative control GST‐His in binding buffer (50 mm Tris, pH 7.5, 200 mm NaCl, 10% glycerol, 0.5% Triton‐X‐100, 10 mg mL⁻¹ RNaseA), supplemented with 1× protease inhibitor complete EDTA‐free (Beyotime Biotechnology). The reaction volume was made upto 300 µL in total and incubated for 1 h at 4 °C. Beads were washed and bound proteins were resolved by SDS‐PAGE and analyzed by western blot.

The full-length cDNA sequence of EjERF39 without stop codon was inserted into the pGEX-4t-1 (GE) vector and introduced into Escherichia coli strain BL21 (Novagen). Expression of the recombinant pGEX-EjERF39 protein in BL21 was fully induced by the addition of IPTG at a final concentration of 1 mM at 16 °C. The target protein was purified following the instructions of the GST-tag Protein Purification Kit (Beyotime), and checked and visualized using SDS-PAGE with Coomassie blue staining. The 40 bp Ej4CL1 promoter probes containing either the wild-type C-repeat/dehydration-responsive element (DRE) (GCCGAC) or a mutated DRE (AAAAAA) were synthesized and 3′ end labeled with biotin (GeneBio), and cold competitor probes were generated without biotinylation. Details of the electrophoretic mobility shift assay (EMSA) experiments were described by Ge et al. (2017) .

LB liquid medium containing ampicillin was inoculated with BL21/pGEX-HpaXcm (pGEX-HpaG-Xcm transformed into BL21 (DE3)). Isopropyl-β-D-thiogalactoside (IPTG) with final concentrations of 0.05 mM and 0.1 mM were added to induce protein expression, until the optical density at 600 nm increased to 0.6–0.8, and then cultured at 28 °C and 37 °C for 3 h and 5 h, respectively. The bacteria collected after centrifugation were resuspended in 1 × PBS (phosphate buffered saline) (Solarbio, Beijing,China) and broken using an ultrasonic wave. After centrifugation, the supernatants and precipitates were collected and identified by performing 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)^{6 (link),25 (link)}. Purified GST-HpaXcm (the fusion protein of a glutathione S-transferase (GST)-tag and HpaG-Xcm) was obtained from crude protein using a GST-tag Protein Purification Kit (Beyotime, Shanghai, China), and GST-HpaXcm was cleaved using thrombin (GE, Boston, MA, USA) at 22 °C for 16 h to obtain the purified HpaG-Xcm. HpaG-Xcm was diluted to 10 μM using PBS before determining the protein concentration by performing a bicinchoninic acid assay (BCA, Solarbio, Beijing, China). GST-HpaXcm was blotted using a polyclonal antibody against GST and a goat anti-rabbit IgG-HRP antibody.

The specific primers (EOnGal8-L-F and EOnGal8-L-R) with restriction sites (BamH I and Xho I) were designed (Table 1) to amplify OnGal8-L ORF. The PCR product was purified and ligated into the pMD18-T vector. The recombinant plasmids pMD-18T-OnGal8-L and pGEX-4T-1 were digested with BamH I and Xho I. The digested products were ligated and transformed into E. coli BL21 (DE3) (TransGen, China). The positive clone was verified by PCR and DNA sequencing. Then, the positive clone was picked to culture in fresh LB liquid medium containing ampicillin (100 μg/mL). When OD₆₀₀ reached 0.4–0.6, isopropyl-β-Dthiogalactopyranpside (IPTG) was added into cultured bacteria with a final concentration of 1 mmol/L. The IPTG added cultures were continuously induced at 37°C for 5 h. The bacteria were collected and washed three times with PBS. Lysozyme was added into bacterial solution with a final concentration of 1 mg/mL, and placed on ice for 30 min, then centrifuged at 4°C for 10 min. The supernatant was purified using a GST-tag protein purification kit (Beyotime, China), desalted and concentrated using an Amicon Ultra Centrifugal Filter (Amicon, USA). The purified protein was analyzed by 10% reducing SDS-PAGE and Western blot. The empty vector (pGEX-4T-1)-expressed GST-tagged protein was prepared as described above and used as control in further analysis.