Hitrap chelating hp ni affinity columns

Fragments of LjGpx1 and LjGpx3 encoding the predicted mature proteins (Supplementary Fig. S1) were amplified by PCR from nodule cDNA using PfuUltra II DNA polymerase (Agilent) and primers (Supplementary Table S1) compatible with pET200 directional TOPO expression kits (Invitrogen). Protein expression was induced in Escherichia coli BL21 (DE3) by the addition of 1mM isopropyl-β-d-thiogalactopyranoside for 4h at 37ºC. Bacteria were harvested by centrifugation, resuspended in 50mM potassium phosphate (pH 8.0) containing 300mM NaCl and 40mM imidazole, and sonicated 6×30 s. Extracts were cleared by centrifugation and supernatants were loaded onto HiTrap chelating HP Ni-affinity columns (GE Healthcare Life Sciences). The His-tagged proteins were eluted with buffer supplemented with 250mM imidazole, desalted, and concentrated by ultrafiltration.

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.