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

Authors: MONA ALHARBI, ABDULRAHMAN MOHAMMAD ALSENAIDY, SOOAD ALDAIHAN, ANWAR AHMED

Abstract: Glutathione reductase (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 (Camelus dromedarius) is unknown. In this study, the mitochondrial glutathione reductase gene (Gsr) from C. dromedarius liver was cloned and highly expressed in Escherichia coli (Jm109) to gain insight into GR functions in the liver. After amplification of the cDNA encoding the functional unit of Gsr (1.2 kb), the products were cloned into the PGEM-T Easy and PET28a vectors. Gsr expression was confirmed using an immunoblotting technique (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 K₃PO₄ buffer. The Michaelis constant, K_m, for the substrates glutathione disulfide (GSSG) and NADPH was 45.6 μM and 63.5 μM, respectively; moreover, maximal velocity (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

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