Characterizing nucleotide binding site domain (NBD) of ZzR1 resistance gene from Zingiber zerumbet: in silico ligand docking and optimizing heterologous expression

Abstract

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 ZzR1, characterized from Zingiber zerumbet 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, pEcoli-Nterm-6xHN and pET Directional TOPO and transformed in five Escherichia coli strains namely DH5α, TOP10, BL21DE3, BL21DE3 star and BL21plysS cells. The NBS protein of 36 kDa molecular size was expressed in E. coli 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.