Gst fusion protein purification kit

To validate the interaction between HN and GRP78, GST and GST-HN recombinant proteins were expressed in Escherichia coli BL-21 cells and purified using the GST Fusion Protein Purification Kit (Genscript, #L00207) as per the manufacturer’s protocol. The Glutathione Sepharose 4B beads (Cytiva, #17075601) were incubated with the purified GST-tagged proteins at 4°C for 8 h, followed by washing with lysis buffer for four times, and further incubated overnight at 4°C with GRP78 protein cell lysates. The interaction complexes were eluted from the beads. The presence of GST, GST-HN and GRP78 was confirmed by Western blotting using mouse anti-GST mAbs and rabbit anti-GRP78 pAbs, respectively.

To validate the interaction between HN and GRP78, GST and GST-HN recombinant proteins were expressed in Escherichia coli BL-21 cells and purified using the GST Fusion Protein Purification Kit (Genscript, #L00207) as per the manufacturer’s protocol. The Glutathione Sepharose 4B beads (Cytiva, #17075601) were incubated with the purified GST-tagged proteins at 4°C for 8 h, followed by washing with lysis buffer for four times, and further incubated overnight at 4°C with GRP78 protein cell lysates. The interaction complexes were eluted from the beads. The presence of GST, GST-HN and GRP78 was confirmed by Western blotting using mouse anti-GST mAbs and rabbit anti-GRP78 pAbs, respectively.

The ORFs of FonAP-2α, FonAP-2β, and FonAP-2μ, and their palmitoylation-deficient variants were cloned into pGEX4T-3 or pET28a. Prokaryotic expression of recombinant proteins in Escherichia coli strain BL-21 was induced by adding IPTG (isopropyl-β-D-thiogalactopyranoside; Sigma-Aldrich, St. Louis, MO, USA) to a final concentration 0.1 mmol/L, and recombinant proteins were purified using a glutathione S-transferase (GST) fusion protein purification kit (Genscript, Nanjing, China) or Profinity IMAC Ni-charged resin (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Cell-free protein degradation assays were carried out following a previously described protocol with modifications (76 (link)). Briefly, total proteins were extracted in degradation buffer (5 mmol/L DTT, 10 mmol/L ATP, 4 mmol/L PMSF, 10 mmol/L MgCl₂, 25 mmol/L Tris-HCl, and 10 mmol/L NaCl) from the WT and ΔFonpat2 strains and then incubated with GST-FonAP-2α, HIS-FonAP-2β, GST-FonAP-2μ, GST-FonAP-2α^C400S, HIS-FonAP-2β^C5,260S, or GST-FonAP-2μ^C24S at 22°C. Protein samples were collected, boiled, separated on 12.5% SDS-PAGE gels, and detected by Western blotting using anti-GST and anti-HIS antibodies (1:1,000 dilution; Genescript). GAPDH was detected with anti-GAPDH antibody and used as a control.

The coding sequences of FonPARP1, FonKin4, and FonKin4-ST were cloned into the pGEX4T-3 vector, featuring a GST tag, or pET32a vector, possessing an HIS tag. Prokaryotic expression and purification of recombinant proteins were performed as previously described (Liu et al., 2019 (link)). Briefly, E. coli Rosetta cells carrying the recombinant vectors were induced by 1 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG; Sigma-Aldrich) at 18°C for 20 h. Subsequently, the recombinant proteins were purified using a GST fusion protein purification kit (Genscript, Piscataway, NJ, United States) and Profinity IMAC Ni-charged resin (Bio-Rad, Hercules, CA, United States), respectively.

The recombinant expression plasmids pCold-SseB and pGEX-6p-1-SseB were transformed into competent E. coli BL21 (DE3) cells. For the expression of rHis-SseB with a His tag at the end of its N-terminuses, E. coli BL21 (DE3) harboring pCold-SseB was induced by 0.5 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) at 15 °C for 24 h. For the expression of rGST-SseB with a GST tag at the end of its N-terminuses, E. coli BL21 (DE3) harboring pGEX-6p-1-SseB was induced by 0.5 mM IPTG at 30 °C for 5 h. The bacteria were harvested and resuspended in ice-cold PBS. The bacterial cells were lysed using ultrasonic tissue homogenizers. The bacterial lysates were evaluated using SDS-PAGE. The soluble recombinant proteins, rHis-SseB and rGST-SseB, were purified using the His•Bind^® Purification Kit (Novagen, San Diego, CA, USA) and GST Fusion Protein Purification Kit (GenScript, Nanjing, China).

RNAiso Plus, First-Strand cDNA Synthesis Kit, and SYBR Premix Ex Taq Polymerase were purchased from TaKaRa Biotech (Dalian, China). TIANamp Marine Animals DNA Kit was obtained from Tiangen Biotech (Beijing, China). GST Fusion Protein Purification Kit and His-tagged Protein Purification Kit were purchased from GenScript (Nanjing, China).