Characterizing nucleotide binding site domain (NBD) of <i>ZzR</i>1 resistance gene from <i>Zingiber zerumbet</i>: <i>in silico</i> ligand docking and optimizing heterologous expression

The Nucleotide-binding site domain (NBD) of plant resistance (R) genes plays a vital role during plant defense signaling. The functional significance of CC-NBS-LRR (Coiled coil-NBS-Leucine Rich Repeat) class of R gene designated <i>ZzR</i>1, characterized from <i>Zingiber zerumbet</i> in earlier studies, was determined by molecular modeling and docking studies. Docked complex showed the ligand GTP interacts with amino acid residues in the cleft made by GLPL and P-loop motifs of the NBD. Heterologous expression of ZzR1 NBS protein was optimized using expression vectors, p<i>Ecoli</i>-Nterm-6xHN and pET Directional TOPO and transformed in five <i>Escherichia coli</i> strains namely DH5α, TOP10, BL21DE3, BL21DE3 star and BL21plysS cells. The NBS protein of 36 kDa molecular size was expressed in <i>E. coli</i> BL21DE3 strain using pET TOPO vector. Optimum induction was detected at 30 °C using isopropyl-1-thio-β-D-galactopyranoside (IPTG) (1 mM). The present study provides valuable information on ligand interactions and heterologous expression of ZzR1 NBD protein.