Overexpression, purification and enzymatic characterization of a recombinant Arabian camel Camelus dromedarius glucose-6-phosphate dehydrogenase

Hesham Saeed; Mohammad Ismaeil; Amira Embaby; Farid Ataya; Ajamaluddin Malik; Manal Shalaby; Sabah El-Banna; Ahmed Abdelrahim Mohamed Ali; Khalid Bassiouny

doi:10.1016/j.pep.2015.09.002

Overexpression, purification and enzymatic characterization of a recombinant Arabian camel Camelus dromedarius glucose-6-phosphate dehydrogenase

Protein Expr Purif. 2018 Feb:142:88-94. doi: 10.1016/j.pep.2015.09.002. Epub 2015 Sep 9.

Authors

Hesham Saeed¹, Mohammad Ismaeil², Amira Embaby³, Farid Ataya⁴, Ajamaluddin Malik², Manal Shalaby⁵, Sabah El-Banna⁶, Ahmed Abdelrahim Mohamed Ali⁷, Khalid Bassiouny⁸

Affiliations

¹ Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt. Electronic address: hesham25166@hotmail.com.
² 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.
³ Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt.
⁴ 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.
⁵ Genetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientific Research and Technology Applications, New Borg Alarab City, Alexandria, Egypt.
⁶ Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt.
⁷ Food and Agricultural Science, Plant Production Department, Biotechnology Laboratory, King Saud University, P.O. Box 2454, Riyadh, Saudi Arabia.
⁸ Molecular Biology Department, Genetic Engineering and Biotechnology Institute, University of Sadat City, Egypt.

PMID: 26363117
DOI: 10.1016/j.pep.2015.09.002

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.

Keywords: Arabian camel; Glucose-6-phosphate dehydrogenase; Reactive oxygen species (ROS).

MeSH terms

Animals
Camelus
Cloning, Molecular
Enzyme Assays
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression
Glucose-6-Phosphate / chemistry*
Glucosephosphate Dehydrogenase / genetics
Glucosephosphate Dehydrogenase / metabolism*
Hydrogen-Ion Concentration
Isoelectric Point
Kinetics
Liver / chemistry
Liver / enzymology
Molecular Weight
NADP / chemistry*
Plasmids / chemistry*
Plasmids / metabolism
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Substrate Specificity

Substances

Recombinant Proteins
NADP
Glucose-6-Phosphate
Glucosephosphate Dehydrogenase