Ni nta agarose affinity beads

The full-length of CCaMK and its site-directed mutants were cloned into the bacterial expression system pET28b and transformed into Escherichia coli strain BL21 (DE3)/pLysS. The bacteria carrying the above plasmids were grown in LB liquid media containing kanamycin at 37°C until OD₆₀₀ of the culture reached 0.5 units. Once the liquid culture reached this optimal density, 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce the recombinant protein. After 3-h induction, cells were harvested and broken using lysozyme treatment (1 mg/ml) followed by sonication. The recombinant protein was purified with Ni-NTA agarose affinity beads (Qiagen) as described in the manufacturer’s manual. The purified proteins were dialyzed against buffer containing 40 mM Tris pH 7.6, 1 mM dithiothreitol (DTT), 1 mM EDTA, and 10% ethylene glycol. Dialyzed proteins were quantified by Bradford assay and stored at -80°C with 15% glycerol.

Gene-specific primers were used to clone the coding sequences of the genes GmCML1, GmCML13, GmCML39, and GmCML95 by PCR from cDNA of soybean seedlings. AtCaM7 were cloned from Arabidopsis cDNA. The gene-specific primers are listed in Supplemental Table S11. Recombinant proteins were expressed and purified according to (Routray et al., 2013 (link)). cDNAs were subcloned into the multiple cloning sites of the pET28a expression vector (Novagen) and confirmed by DNA sequencing. Recombinant proteins were expressed in Escherichia coli BL21(DE3) pLysS (Stratagene). Bacterial cultures were grown under antibiotic selection in Luria-Bertani medium with agitation at 37°C to an OD600 of 0.4–0.5 and protein expression was induced over the course of 3 h by the addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). His-tagged recombinant proteins were purified using Ni-NTA agarose affinity beads (Qiagen) as described by the manufacturer. The electrophoresis mobility-shift assay was performed as described (Garrigos et al., 1991 (link)) using 1–2 μg of denatured protein supplemented with either 1.0 mM CaCl₂, 1.0 mM EGTA, or 1.0 mM MgCl₂. Each sample was electrophoresed on a 12% SDS–PAGE containing either 1.0 mM CaCl₂, 1.0 mM EGTA, or 1.0 mM MgCl₂, respectively.

The E. coli strain BL21(DE3)/pLysS carrying the above pET32a-derived plasmids for expression of recombinant proteins of the wild-type versions of AtSR1 were obtained from a previous project. The E. coli strain was cultured in Super Optimal broth with Catabolite repression (SOC) media for 3 h with the concentration of OD₆₀₀ = 0.5. Then, Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added with a final concentration of 0.5 mM IPTG for 3 h. 6His-tagged recombinant proteins were purified using Ni-NTA agarose affinity beads (Qiagen) as described by the manufacturer [63 (link),64 (link)]. Recombinant AtSR1 covering CG-1 domain was used for EMSA to detect its interaction with the promoter fragments of target genes.