Recombinant prset plasmid

The recombinant pRSET plasmid (Invitrogen) containing the 1086 bp CDS of SAPK9 in XhoI and EcoRI restriction sites allowed expression of 6xHis N-terminal tagged SAPK9 protein in E. coli BL21 (DE3) pLysE strain (Invitrogen) upon 1 mM IPTG induction. Similarly, first 900 bp (encoding 300-amino acid) from the OsbZIP23 CDS of O. rufipogon [25 (link)] was cloned into a pRSET vector in BamHI and EcoRI restriction sites, transformed and expressed in pLysE cells with 0.5 mM IPTG induction. The expressed proteins were purified in native condition and used for in vitro kinase assay. In vitro phosphorylation of generic substrate histone III (Sigma) was performed as described previously [28 (link)]. In vitro phosphorylation of OsbZIP23 was performed by incubating the individual reaction mixture for 5, 25 and 40 min at 25 °C following the above-mentioned protocol. The products were fractionated in 12 % SDS-PAGE and visualized by autoradiography.

The in vitro kinase assay was performed according to the methods described by Dey et al. (2016) (link). The recombinant pRSET plasmid (Invitrogen) containing the 1086 bp coding sequence (CDS) of OsSAPK9 in XhoI and EcoRI restriction sites allowed expression of 6×His N-terminal tagged OsSAPK9 protein in Escherichia coli BL21 (DE3) pLysE strain (Invitrogen) upon induction by 1 mM isopropyl-β-d-thiogalactopyransode (IPTG). Similarly, first the 893 bp (encoding 297 amino acids) from the OsbZIP20 CDS of Oryza rufipogon were cloned into a pRSET vector in BamHI and EcoRI restriction sites, transformed, and expressed in pLysE cells with induction by 0.5 mM IPTG. The expressed proteins were purified in the native condition and used for the in vitro kinase assay. In vitro phosphorylation of the generic substrate histone III (Sigma) was performed as described previously. In vitro phosphorylation of OsbZIP20 was performed by incubating the individual reaction mixture for 40 min at 25 °C following the above-mentioned protocol. The products were fractionated by 12% SDS–PAGE and visualized by autoradiography.