Protein expression and purification

The rPfGDH clone was grown at 37 °C in 1 l Terrific broth containing 0.5% glucose (Himedia, India) supplemented with 100 μg/ml ampicillin to an OD₆₅₀ value of 0.4–0.6, was induced with 1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) and the culture was further incubated for 6 h at 37 °C at 225 rpm. Subsequently, the culture was centrifuged at 8000 g for 10 min to harvest the cells and stored at −20 °C. Later, cell pellet was resuspended in 20 ml GenePro Total Protein isolation buffer (Genetix, India) and incubated on ice for 10 min and centrifuged at 16000 g for 10 min. The clear cell lysate was collected and mixed with 1.5 M GuHCl and passed through 2 ml bed volume of cOmplete His-Tag purification resin (Roche, USA) using gravity flow columns (Biorad, USA). The column was washed with 4 bed volumes of buffer containing 40 Tris-HCl pH 7.4 and 20 mM and 50 mM Imidazole. rPfGDH was eluted with 100 and 150 mM imidazole, both eluates were mixed and passed through ultra-centrifugal device with a cut-off of 30 Kda (Ultracell, Merck, Millipore) at 5000 g for 10 min at 4 °C. The purification procedure was monitored by SDS-PAGE denaturing conditions, where the rPfGDH was appeared approximately as a ~52 kDa, which was in the agreement with its theoretical molecular mass (Fig. 2C). The rPfGDH was confirmed by his-tag monoclonal antibodies, mouse (Puregene, Genetix, India) and goat-anti mouse IgG-HRP antibodies on western blot using Biorad Mini-PROTEAN Tetracell system (Fig. 2D). The rPfGDH protein concentration estimated was 4 mg/ml against BSA standard.