Gst resin

To produce glutathione S-transferase (GST)-KFB20 and GST-KFB39, the full-length coding sequences of KFB20 and KFB39 were separately amplified and cloned into the pGEX-4T-1 vector. To produce PAL1-His, the coding sequence of PAL1 was amplified and cloned into the pET-28a (+) vector. Protein expression was induced in the Escherichia coli BL21 (DE3) cell line by 0.3 mM IPTG and expressed GST fusion protein was purified using GST resin (C600031, Sangon Biotech, China) and expressed His fusion protein was purified using Ni-NTA agarose (30210, Qiagen, Germany).
To test the influence of CA on the interaction between PAL and KFB proteins, the GST pull-down assay was performed, and 140 μg GST-KFB20 or GST-KFB39 with 100 μg PAL1-His were added into the 1 mL reaction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM Na₂EDTA, 10% glycerol, 0.1% Triton X-100, 1 × complete protease inhibitor cocktail, 1 mM DTT) containing different concentrations of CA, respectively. GST resin was used to bind GST-KFB20 or GST-KFB39. Proteins captured by GST resin were detected by immunoblotting with anti-His antibody (D410002, Sangon Biotech, China) and the Ponceau S staining method.

In the experiment on prokaryotic recombinant expression, primers (HepCL-EX-F/R, HepCL-N-EX-F/R, HepCL-C-EX-F/R, Table 1) were used to amplify fragments of HepCL (957 bp), HepCL-N (345 bp), and HepCL-C (519 bp). PCR was programmed at 95°C for 5 min, 35 cycles at 95°C for 30 s, 58°C for 30 s, 72°C for 50 s, and one cycle at 72°C for 10 min. The DNA fragments were linked to the vector pGEX-4T-1. Recombinant HepCL, HepCL-N, and HepCL-C were expressed in Escherichia coli (E. coli) BL21 cells (TransGen, China). The recombinant proteins were purified by affinity chromatography using GST resin (Sangon, China) following the method described in previous papers (38 (link)). HepCL polyclonal antibodies were prepared by the company using recombinant proteins (Frdbio, China).

We cloned GmWAK1 into the pET29b (+) expression vector and GmANNRJ4 into the pGEX-4T-1 expression vector. His-GmWAK1 and glutathione S-transferase (GST)-GmANNRJ4 proteins were separately produced in E. coli BL21 (DE3) cells, which we then harvested and purified using a GST-Sefinose kit (Sangon, China, code number C590927) or a His-bind Purification Kit (Merck Millipore, Burlington, MA, USA, code number 70239-3). We performed the pull-down assay as described by Yang et al. [94 (link)], with minor modifications. In a total volume of 1 mL of GST binding buffer (Sangon), we incubated the GST or GmANNRJ4-GST recombinant proteins for 1 h at 4 °C with 400 mL of GST resin (Sangon), after which we added an equal volume of the GmWAK1-His recombinant protein and then incubated for 6 h at 4 °C. We washed the binding reaction five times with binding buffer, each for 10 min at 4 °C; then, we eluted the pulled-down proteins by boiling, which we separated on a 12% SDS-PAGE gel and immunoblotted with anti-His antibody and anti-GST antibody (Abmart, Berkeley Heights, NJ, USA; part numbers M20020 and M20025).