논문 상세정보
Overexpression, purification and enzymatic characterization of a recombinant Arabian camel Camelus dromedarius glucose-6-phosphate dehydrogenase
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초록
Abstract In a previous study the full-length open reading frame of the Arabian camel, Camelus dromedarius liver cytosolic glucose-6-phosphate dehydrogenase (G6PD) cDNA was determined using reverse transcription polymerase chain reaction. The C. dromedarius cDNA was found to be 1545 nucleotides (accession number JN098421) that encodes a protein of 515 amino acids residues. In the present study, C. dromedarius recombinant G6PD was heterologously overexpressed in Escherichia coli BL21 (DE3) pLysS and purified by immobilized metal affinity fast protein liquid chromatography (FPLC) in a single step. The purity and molecular weight of the enzyme were analyzed on SDS–PAGE and the purified enzyme showed a single band on the gel with a molecular weight of 63.0 KDa. The specific activity was determined to be 2000 EU/mg protein. The optimum temperature and pH were found to be 60 °C and 7.4, respectively. The isoelectric point (pI) for the purified G6PD was determined to be 6.4. The apparent K m values for the two substrates NADP + and G6P were found to be 23.2 μM and 66.7 μM, respectively. The far-UV circular dichroism (CD) spectra of G6PD showed that it has two minima at 208 and 222 nm as well as maxima at 193 nm which is characteristic of high content of α-helix. Moreover, the far-UV CD spectra of the G6PD in the presence or absence of NADP + were nearly identical. Highlights C. dromedarius recombinant G6PD was overexpressed in E. coli BL21 (DE3) pLysS and purified by affinity fast protein liquid chromatography (FPLC). Recombinant G6PD was found to be immunoreacted with anti-human G6PD antibody. The pI was found to 6.4 Analyses of CD spectrum of camel G6PD showed that it has two minima at 208 and 222 nm as well as maxima at 193 nm which is a characteristic of high content of α-helix. NADP + binding did not notable alter the secondary structures.
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주제어
Arabian camel . Glucose-6-phosphate dehydrogenase . Reactive oxygen species (ROS).
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