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

In this study, the kinase domain of Nicotiana tabacum GCN2 (NtGCN2) was inserted into the pET15b vector for prokaryotic expressionin 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 Ni2+-NTA agarose column, anion exchange, and molecular sieve, and the purified proteinwas 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. Our study has prepared a specific antibody, and proves NtGCN2 can phosphorylate NteIF2α in vitro, which lays a foundation for further study of the function and interaction network of NtGCN2.
Source: Protein Expression and Purification - Category: Biochemistry Source Type: research