Recombinant Protein Expression and Purification

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.

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Wang J., Gao Y., Xiong X., Yan Y., Lou J., Noman M., Li D, & Song F. (2024). The Ser/Thr protein kinase FonKin4-poly(ADP-ribose) polymerase FonPARP1 phosphorylation cascade is required for the pathogenicity of watermelon fusarium wilt fungus Fusarium oxysporum f. sp. niveum. Frontiers in Microbiology, 15, 1397688.

Publication 2024

Isopropyl ß-D-1-thiogalactopyranoside (IPTG; GST fusion protein purification kit Profinity IMAC Ni-charged resin

Corresponding Organization :

Other organizations : Zhejiang University, ZheJiang Academy of Agricultural Sciences, Institute of Plant Protection

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Variable analysis

independent variables

Cloning the coding sequences of FonPARP1, FonKin4, and FonKin4-ST into pGEX4T-3 vector with GST tag and pET32a vector with HIS tag
IPTG induction of E. coli Rosetta cells carrying the recombinant vectors at 18°C for 20 h

dependent variables

Prokaryotic expression and purification of recombinant proteins

control variables

Purification methods used: GST fusion protein purification kit and Profinity IMAC Ni-charged resin

controls

No positive or negative controls were explicitly mentioned in the provided information.

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