title: Overexpression, purification and enzymatic characterization of a recombinant Arabian camel Camelus dromedarius glucose-6-phosphate dehydrogenase.
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 Kmvalues 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.
authors.affiliation: Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt. Electronic address: hesham25166@hotmail.com.
authors.forename: Hesham
authors.initials: H
authors.lastname: Saeed
authors.affiliation: Department of Biochemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; Protein Research Chair, Biochemistry Department, College of Science King Saud University, Bld. 5, P.O. Box 2454, Riyadh, Saudi Arabia.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Ismaeil
authors.affiliation: Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt.
authors.forename: Amira
authors.initials: A
authors.lastname: Embaby
authors.affiliation: Department of Biochemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; Molecular Biology Department, Genetic Engineering Division, National Research Centre, 33 El-Bohouth St. (former El-Tahrir St.), P.O. 12622, Dokki, Giza, Egypt.
authors.forename: Farid
authors.initials: F
authors.lastname: Ataya
authors.affiliation: Department of Biochemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; Protein Research Chair, Biochemistry Department, College of Science King Saud University, Bld. 5, P.O. Box 2454, Riyadh, Saudi Arabia.
authors.forename: Ajamaluddin
authors.initials: A
authors.lastname: Malik
authors.affiliation: Genetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientific Research and Technology Applications, New Borg Alarab City, Alexandria, Egypt.
authors.forename: Manal
authors.initials: M
authors.lastname: Shalaby
authors.affiliation: Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt.
authors.forename: Sabah
authors.initials: S
authors.lastname: El-Banna
authors.affiliation: Food and Agricultural Science, Plant Production Department, Biotechnology Laboratory, King Saud University, P.O. Box 2454, Riyadh, Saudi Arabia.
authors.forename: Ahmed Abdelrahim Mohamed
authors.initials: AAM
authors.lastname: Ali
authors.affiliation: Molecular Biology Department, Genetic Engineering and Biotechnology Institute, University of Sadat City, Egypt.
authors.forename: Khalid
authors.initials: K
authors.lastname: Bassiouny
mesh_terms: D000818:Animals; D002162:Camelus; D003001:Cloning, Molecular; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D019298:Glucose-6-Phosphate / Q000737:chemistry*; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D008970:Molecular Weight; D009249:NADP / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D009249:NADP; D019298:Glucose-6-Phosphate; D005954:Glucosephosphate Dehydrogenase
keywords: Arabian camel; Glucose-6-phosphate dehydrogenase; Reactive oxygen species (ROS)