Expression, purification, and in vitro characterization of kinase domain of NtGCN2 from tobacco

Abstract General control nonderepressible 2 (GCN2) can phosphorylate the α subunit of eukaryotic initiation factor eIF2 (eukaryotic translation initiation factor 2) to down-regulate protein synthesis in response to various biotic and abiotic stresses. However, the kinase activity of plant GCN2 has not been well-characterized in vitro . In this study, the kinase domain of Nicotiana tabacum GCN2 (NtGCN2) was inserted into the pET15b vector for prokaryotic expression in Escherichia coli BL21-CodonPlus-(DE3)- RIPL after induction by 0.5 mmol L −1 IPTG for 13 h at 16 °C. The soluble protein was collected and purified by Ni 2+ -NTA agarose column, anion exchange, and molecular sieve, and the purified protein was used for kinase assays and the preparation of a polyclonal antibody. Enzyme-linked immunosorbent assay results showed that the titer of the antiserum was 1:520K. Western blot analysis showed that the prepared antibody reacted with GCN2 in tobacco. Additionally, the kinase activity of NtGCN2 was characterized by using recombinant NteIF2α protein as a substrate in vitro . The results showed that NtGCN2 phosphorylated NteIF2α in vitro , with the level of phosphorylation positively correlated with the NtGCN2 concentration and reaction time. The results provide basic data for further studies to understand the function of plant GCN2 and GCN2-mediated stress response network.