Generation of polyclonal antibodies against recombinant Agrobacterium tumefaciens decaprenyl diphosphate synthase produced in Escherichia coli

Coenzyme Q (CoQ), an electron carrier of the mitochondrial respiratory chain and a potent antioxidant, consists of a benzoquinone head and an isoprenoid side chain of varying numbers of isoprene units, 10 in humans, animals and some microbes including Agrobacterium tumefaciens (CoQ10), 9 in Arabidopsis and cereals (CoQ9) and 8 in Escherichia coli (CoQ8). The synthesis of CoQ10 side chain is catalyzed by a single side chain length determining enzyme, decaprenyl diphosphate synthase (DPS) which is encoded by a dps gene. We report here, the isolation and sequencing of a dps gene from A. tumefaciens strain LBA4404 and its cloning in pET28a expression vector of E. coli BL21 (DE3) strain to produce and purify recombinant DPS protein for the production of its antibodies in rabbit. The dps gene isolated from A. tumefaciens consists of 1077 bp efficient for encoding a polypeptide of 359 amino acids with a molecular mass of around 39 kDa. DPS protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside in bacterial culture to a final concentration of 1.0 mM. Recombinant protein tagged with histidine at N-terminal was purified by immobilized metal-affinity chromatography. The purified DPS protein was capable of producing polyclonal antibodies in rabbit whose specificity to DPS enzyme was confirmed by the western blot analysis. Expression of dps gene in E. coli extended the side chain length of CoQ8 to 10 isopenoid units and produced CoQ10 in addition to CoQ8. This is the first report on the production of polyclonal antibodies against recombinant DPS protein and demonstration of their utility.