Purification of Plant Antioxidant Enzymes

Expression and purification of cowpea (Vigna unguiculata) iron-superoxide dismutase (VuFeSOD; GeneBank AAF28773.1) and Lotus japonicus glutathione peroxidase (LjGpx3; Lotus Base: LotjaGi4g1v0458000) were carried out as described by Moran et al. (2003) (link) and Matamoros et al. (2015) (link), respectively. Briefly, PCR fragments encoding the mature VuFeSOD and LjGpx3 proteins were cloned into pET-28a(+) (Novagen) and pET200/D-TOPO, respectively. His-tagged proteins were produced in Escherichia coli BL21 (DE3), purified on HiTrap chelating HP Ni-affinity columns (GE Healthcare Life Sciences), desalted, and concentrated by ultrafiltration. L. japonicus phytoglobin (LjGlb3-1; Lotus Base: Lj1g3v2035270.1) was cloned into pET11a (Novagen) with an N-terminal Strep-tag and was expressed in E. coli C41(DE3) cells (Lucigen) as described (Villar et al., 2018 ). The Strep-tagged protein was purified on a StrepTactin Sepharose High Performance column (GE Healthcare), desalted, and concentrated by ultrafiltration.

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Matamoros M.A., Romero L.C., Tian T., Román Á., Duanmu D, & Becana M. (2023). Persulfidation of plant and bacteroid proteins is involved in legume nodule development and senescence. Journal of Experimental Botany, 75(10), 3009-3025.

Publication 2023

HiTrap chelating HP Ni-affinity columns StrepTactin Sepharose High Performance column

Corresponding Organization : Consejo Superior de Investigaciones Científicas

Other organizations : Universidad de Sevilla, Instituto de Bioquímica Vegetal y Fotosíntesis, Huazhong Agricultural University

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Variable analysis

independent variables

Expression and purification of cowpea (Vigna unguiculata) iron-superoxide dismutase (VuFeSOD; GeneBank AAF28773.1) and Lotus japonicus glutathione peroxidase (LjGpx3; Lotus Base: LotjaGi4g1v0458000)

dependent variables

Amounts or concentrations of purified VuFeSOD and LjGpx3 proteins

control variables

Protocols for expression and purification of VuFeSOD and LjGpx3 as described by Moran et al. (2003) and Matamoros et al. (2015), respectively
Use of pET-28a(+) and pET200/D-TOPO vectors for cloning and expression of VuFeSOD and LjGpx3, respectively
Use of E. coli BL21 (DE3) cells for expression of VuFeSOD and LjGpx3
Purification of His-tagged VuFeSOD and LjGpx3 proteins on HiTrap chelating HP Ni-affinity columns
Desalting and concentration of VuFeSOD and LjGpx3 proteins by ultrafiltration
Cloning of L. japonicus phytoglobin (LjGlb3-1; Lotus Base: Lj1g3v2035270.1) into pET11a vector with an N-terminal Strep-tag
Expression of LjGlb3-1 in E. coli C41(DE3) cells
Purification of Strep-tagged LjGlb3-1 protein on a StrepTactin Sepharose High Performance column
Desalting and concentration of LjGlb3-1 protein by ultrafiltration

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