Gene cloning and expression analysis of mitochondrial glutathione reductase from Arabian camel ( Camelus dromedarius ) liver in Escherichia coli

Authors : Mona ALHARBI, Abdulrahman ALSENAIDY, Sooad ALDAIHAN, Anwar AHMED

Pages : 656-666

View : 17 | Download : 7

Publication Date : 0000-00-00

Article Type : Research Paper

Abstract :Glutathione reductase insert ignore into journalissuearticles values(GR); is highly conserved among diverse taxa and has important biochemical functions. These functions may facilitate survival in harsh conditions, but the role of GR from the liver of the Arabian camel insert ignore into journalissuearticles values( Camelus dromedarius ); is unknown . In this study, the mitochondrial glutathione reductase gene insert ignore into journalissuearticles values( Gsr ); from C. dromedarius liver was cloned and highly expressed in Escherichia coli insert ignore into journalissuearticles values(Jm109); to gain insight into GR functions in the liver. After amplification of the cDNA encoding the functional unit of Gsr insert ignore into journalissuearticles values(1.2 kb);, the products were cloned into the PGEM-T Easy and PET28a vectors. Gsr expression was confirmed using an immunoblotting technique insert ignore into journalissuearticles values(45 kDa);. Recombinant GR was purified to homogeneity using Ni-NTA resins, with an overall yield of 7.23% and a specific activity of 0.3063 U/mg. The optimum pH of recombinant GR was 7 and the optimum temperature was 35 °C in 50 mM K 3 PO 4 buffer. The Michaelis constant, K m , for the substrates glutathione disulfide insert ignore into journalissuearticles values(GSSG); and NADPH was 45.6 μM and 63.5 μM, respectively; moreover, maximal velocity insert ignore into journalissuearticles values(V max ); values were 3.969 x 10 -2 U/mg and 1.497 x 10 -1 U/mg. This partial characterization of camel liver GR extends our insight into the ability of camels to cope with harsh environmental conditions.
Keywords : Camelus dromedaries liver, pGEM T Easy vector, Ni NTA resin, recombinant glutathione reductase, partial characterization