Glutathione S-Transferase Pull-Down Assays

The pGEX-4T-1 (CW biotech, Peking, China) vector was digested by BamH I and Xho I (TaKaRa). The full CDS fragments for the SBP1 and SSK1 genes were amplified by PCR experiments. Using BamH I and Xho I, the PCR products were digested and then cloned into the digested pGEX-4T-1 vector, respectively, to produce GST fusion proteins. For producing His₆-tagged fusion proteins, the full CDSs of CUL1 and SFB17 were cloned into the pET-32a vector (EMD Biosciences, Novagen) at BamH I and Xho I (Takara), while the full CDS of SFB16 was cloned into the pET-32a vector at BamH I and Hind III (Takara). The used primers were as follows: SBP1: 5′-CGG GAT CCA TGG CTC TTC CTC AAC ACC ACT TTC-3′ (forward), 5′-CCG CTC GAG CAA ATA TAC CTC CAT GCC G-3′ (reverse); SSK1: 5′-CGG GAT CCA TGT CGG CCG AGG AGG AGA AGA AC-3′ (forward), 5′-CCG CTC GAG GTC CTC ATC AAC TCC TTC-3′ (reverse); CUL1: 5′-CCG CTC GAG ATG GAA CGG AAA ATT ATT GAG-3′ (forward), 5′-TTA CGG GAT CCT GCA AGA TAC TTG AAC AT-3′ (reverse); SFB16: 5′-CGG GAT CCA TGA TTT TCT TCA GGA TGA CAT TC-3′ (forward), 5′-CCG CTC GAG ATA ATT CTT GAA TAA AAC CAA AC-3′ (reverse); SFB17: 5′-CGG GAT CCA TGA ATT TCG ATA GTC CTA TAC AC-3′ (forward), 5′-CCG CTC GAG CCC GAT TGT ACG ATT ATA ATA ATC-3′ (reverse).

All the constructed vectors were transformed into Escherichia coli strain (BL21), and the transformed cells were cultured in LB medium containing 60 µg/ml ampicillin at 16°C with shaking at 220 rpm. When the OD₆₀₀ achieved 0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added into the suspension culture to induce protein expression, with the conditions being 22°C for 14 h and 0.2 mM IPTG.

To gather bacteria, centrifugation was carried out, and a PBS buffer was used for suspending the bacteria. Subsequently, an ultrasonic cell disruptor was set at 200 W for crushing, and the ultrasonic frequency worked for 10 s, resting for 8 s for 30 min. The GST-tagged fusion proteins were purified using Glutathione Sepharose 4B (Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) and eluted with buffer comprising 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 3 mM DTT, and 15 mM reduced glutathione. The His₆-tagged fusion proteins were purified using Ni-NTA (EMD Biosciences, Novagen) and eluted with buffer comprising 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 250 mM imidazole. All the eluted proteins were dialyzed with buffer composed of 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 3 mM DTT (wash buffer).

For GST pull-down assays, equal amounts of GST-tagged fusion protein and His₆-tagged fusion protein were mixed and incubated on ice for 3 h. Subsequently, we loaded the mixture onto Glutathione Sepharose 4B resin columns. After washing five times with buffer, the proteins were eluted with wash buffer supplemented with 15 mM reduced glutathione. The eluates were separated using 12% SDS-PAGE, and then they were transferred into PVDF membranes (Millipore, Billerica, MA, USA) and probed with anti-His (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). GST and His₆ from GensCript (Nanjing, China) were used as negative controls. There were three replications for each pull-down assay.