Fig. 5

Heterologous expression of NpNBS in Escherichia coli. a Agarose gel electrophoresis of PCR amplified candidate NpNBS from a N. pseudonarcissus cDNA library. Product size is 560 bp (ORF 492 bp + Gateway adapters 68 bp). Numbers on the left refer to the location of standard DNA molecular markers in bp. b Agarose gel electrophoresis of PCR amplified NpNBS performed with 5 positively transformed bacterial colonies (col.1 to col.5) shows a band of NpNBS ORF transcript size of 492 bp, L is for Ladder; c SDS-PAGE analysis of NpNBS protein. Purified protein was extracted from 0.9 mM IPTG induced E. coli Rosetta™ (DE3) pLysS host strain at 37 °C for 8 h in elution 1 with 300 mM of imidazole. L-Ladder; +C- positive control with a 30 kDa protein; NI-non-induced protein; L1- lysate 1 (10 mM imidazole); L2- lysate 2 (10 mM imidazole); W1-wash 1 (20 mM imidazole); W2-wash 2 (20 mM imidazole); E1-elution 1 (300 mM imidazole); E2-elution 2 (300 mM imidazole). d Western blot analysis performed using 6x HIS-tag epitope antibody shows expression of recombinant protein at 37 °C in 0.9 mM IPTG induced crude and pure protein extracts. No detectable expression of recombinant protein at 25 °C in 0.9 mM IPTG induced crude and pure protein extracts. Lane L-ladder; NI- protein non-induced; +C-positive control protein with C-terminal His tags 30 kDa; Cr-crude protein 37 °C/8 h/0.9 mM; E1- elution 1, 37 °C/8 h/0.9 mM; E2- elution 2, 37 °C/8 h/0.9 mM; Cr-crude 25 °C/8 h/0.9 mM; E1- elution 1, 25 °C/8 h/0.9 mM; E2-elution 2, 25 °C/8 h/0.9 mM. Numbers on the left refer to the location of standard protein molecular weight markers in kDa