Accepted Manuscript

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

Hesham Saeed, Mohammad Ismaeil, Amira Embaby, Farid Shokry Ataya,Ajamaluddin Malik, Manal Shalaby, Sabah El-Banna, Ahmed AbdelrahimMohamed Ali, Khalid Bassiouny

PII: S1046-5928(15)30052-8DOI: http://dx.doi.org/10.1016/j.pep.2015.09.002Reference: YPREP 4777

To appear in: Protein Expression and Purification

Received Date: 13 June 2015Revised Date: 27 August 2015Accepted Date: 3 September 2015

Please cite this article as: H. Saeed, M. Ismaeil, A. Embaby, F. Shokry Ataya, A. Malik, M. Shalaby, S. El-Banna,A. Abdelrahim Mohamed Ali, K. Bassiouny, Overexpression, purification and enzymatic characterization of arecombinant Arabian camel Camelus dromedarius glucose-6-phosphate dehydrogenase, Protein Expression andPurification (2015), doi: http://dx.doi.org/10.1016/j.pep.2015.09.002

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Overexpression, purification and enzymatic characterization of a recombinant Arabian

camel Camelus dromedarius glucose-6-phosphate dehydrogenase

Hesham Saeed1, Mohammad Ismaeil

2, 3 Amira Embaby

1, Farid Shokry Ataya

2, 4,

Ajamaluddin Malik2, 3

, Manal Shalaby5, Sabah El-Banna

6, Ahmed Abdelrahim

Mohamed Ali 7 and Khalid Bassiouny

8

1* Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria

University, Alexandria, Egypt.

2 Biochemistry Department, College of Science King Saud University, Bld. 5, Lab AA10,

P.O. Box: 2454 Riyadh. KSA.

3 Protein Research Chair, Biochemistry Department, College of Science King Saud

University, Bld.5, P.O.Box:2454 Riyadh. KSA.

4 National Research Centre, 33 El-Bohouth St. (former El-Tahrir St.), Dokki, Giza, Egypt,

P.O. 12622, Affiliation ID: 60014618.

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. KSA.

8 Molecular Biology Department, Genetic Engineering and Biotechnology Institute,

University of Sadat City, Egypt

*Author to whom correspondence should be addressed; E-Mail: hesham25166@hotmail.com;

hsaeed1@ksu.edu.sa; Tel.: 00966590236357; Fax: 00966124675791.

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, Camelus dromedarius recombinant G6PD was

heterologously overexpressed in Eschericia 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 analysed 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 Km 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).

Abbreviations:

Circular Dichroism (CD); G6PD; glucose-6-phosphate dehydrogenase; G6P, glucose-6-

phosphate; TB, terrific broth; IPTG, isopropyl-thio-β-D-galactopyranoside; NADP+,

nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine

dinucleotide phosphate; NBT, nitroblue tetrazolium; SDS-PAGE, sodium dodecyl sulphate

polyacrylamide gel electrophoresis; ROS, reactive oxygen species.

Introduction

Glucose 6-phosphate dehydrogenase (G6PD, E.C. 1.1.1.49; β-D-glucose-6-phosphate; NADP

oxidoreductase) is one of the housekeeping enzymes that catalyses the rate-limiting reaction

in the pentose phosphate pathway (PPP), providing reducing power to all cells in the form of

NADPH (reduced form of nictotinamide adenine dinucleotide phosphate) and ribose 5-

phosphate sugar for the synthesis of nucleic acids [1]. G6PD catalyses the conversion of

glucose 6-phosphate to 6-phosphoglucono-δ-lactone with concomitant reduction of NADP+

to NADPH. NADPH enables cells to counterbalance oxidative stress that can be triggered by

several oxidant agents, and to preserve the reduced form of glutathione which is essential for

the reduction of hydrogen peroxide and oxygen radicals and the maintenance of red blood

cell proteins especially haemoglobin in the reduced state [2, 3]. NADPH serves also as a

hydrogen and electron donor for a variety of reductive reactions including biosynthesis of

fatty acids, cholesterol and some amino acids [4, 5]. G6PD is found in the cytosol and in

peroxisomes of cells and widely distributed among microorganisms, plants and in different

animal tissues [6, 7]. Recently, G6PD has been revealed to be involved in apoptosis,

angiogenesis and the efficacy to anti-cancer therapy, making it as a promising target in cancer

therapy as one of the final products of the PPP, ribose-5-phosphate, is necessary for nucleic

acid synthesis and tumour progression [8, 9]. Furthermore, several studies described

significant roles of G6PD during abiotic and biotic stress in higher plants [10-12]. At the

molecular level, G6PD has been well studied in many eutherian species including human,

mouse, rats and plants [4, 12-16]. The domesticated one-humped Arabian camel, C.

dromedarius, is one of the most important animals in the Arabian Peninsula. The Arabian

camel is able to live in the harsh desert conditions such as high temperature, direct exposure

to sunlight and without drinking water for weeks [17]. Despite being an essential source of

food in many parts of the Arabian Peninsula, the one-humped camel is the least studied of all

domestic species of mammals [18]. The Arabian camel are continuously exposed to toxic

harmful agents from the environment and from different metabolic processes that produce

reactive oxygen species (ROS). If not eliminated, ROS exerts damage to DNA and proteins

and therefore pose a major threat to the genetic integrity of cells [19]. Camel G6PD is an

important cytosolic enzyme involved in carbohydrate metabolism and generating NADPH

that consumed in the lipogenesis process and in the elimination of reactive oxygen species

(ROS) through different metabolic pathways that involved NADPH as a coenzyme. Hence,

G6PD deficiency impairs the ability of some cells such as erythrocytes to form NADPH

resulting in hemolysis and anemia [2, 19]. In this study we reported overexpression of

recombinant C. dromedarius G6PD in E.coli BL21 (DE3) pLysS and its subsequent

purification and characterization. Moreover, the structural features of His-tagged G6PD were

evaluated by Circular dichroism.

Materials and methods

Chemicals

All chemicals used were of analytical reagent, molecular biology, or chromatographic grade

as appropriate. Water was deionized and distilled.

Bacterial strain and plasmid

E.coli BL21 (DE3) pLysS (Promega, USA) was used as an expression host for over-

production of the recombinant C. dromedarius full-length cDNA cloned into pET28a (+)

(Novagen Co.) plasmid in unique EcoRI and HindIII sites [2]. Expression of the cloned

G6PD is under the control of T7 promoter.

Production, expression and purification of recombinant C. dromedarius G6PD

A 5 L Erlenmeyer flask containing 1000 ml of Terrific broth supplemented with Kanamycin

and Chloramphenicol at a concentration of 50 and 35 µg/ml respectively were inoculated

with 10 ml of an overnight culture of E. coli BL21 (DE3) pLysS pET28a (+) carrying the

G6PD cDNA. The flasks were shaken at 250 rpm at 37 °C until the optical density at 600 nm

was 0.8 at which point IPTG was added at a concentration of 2 mM to the culture and the

temperature was shifted to 30 °C and 150 rpm for 18 hours. Samples (2 ml) were taken at

different time intervals to monitor the expression of the recombinant G6PD. After the period

of incubation the cells were collected by centrifugation at 8000 rpm at 4 °C for 20 min,

washed with 50 mM potassium phosphate buffer pH 7.5 containing 5.0 mM MgCl2 and

pelleted for a second time under the same conditions. The cells were then resuspended in 20

ml 50 mM potassium phosphate buffer containing 5.0 mM MgCl2, 0.2 mM DTT and 10%

glycerol (buffer A) and sonicated using 4X 15 seconds pulses. After the first pulse 1 mM

PMSF, 0.1 µg/ml leupeptin and 0.04 U/ml aprotonin were added to the tubes and then

sonication was continued. After, cells debris were removed by centrifugation at 12,000 rpm at

4 °C for 10 min after which the supernatant was collected for enzyme and protein assays and

for purification experiment.

Enzyme assay and kinetics study

Assay of recombinant G6PD was determined spectrophotometrically by monitoring the

NADPH production at 340 nm and at 25°C [2]. The assay mixture contained 10 mM MgCl2,

0.2 mM NADP+ and 0.6 mM G-6-P in 50mM potassium phosphate buffer, pH7.5. Assay was

carried out in duplicate and the activity was followed for 60s. One unit of G6PD activity is

the amount of enzyme required to reduce one µmole of NADP+ per minute under the assay

conditions. Specific activity is defined as units per mg of protein and fold purification was

calculated by dividing the specific activity of the specified step by the specific activity of the

initial step.

Substrate kinetics were determined at 37 °C in 1 ml reaction mixture containing 50 mM

potassium phosphate buffer, pH 7.5, 10 mM MgCl2 and various concentrations of NADP+

and G6P. A matrix of substrate and coenzyme concentrations between 20-300 µM (NADP+)

and 10-200 µM (G6P) was formed and the reactions were initiated by the addition of enzyme.

The activities were used in constructing Lineweaver-Burk and additional diagnostic plots to

calculate Km values for substrate and coenzyme and Ki values for the inhibitor, NADPH.

The kinetic data were analysed and kinetic constants were determined from the Lineweaver-

Burk plot using GraphPad Prism the non-linear curve-fitting program (version 5.00 for

Windows, GraphPad Software, San Diego, CA). Inhibition by NADPH was performed by

changing the concentrations of NADPH in the assay mixture in presence of varying the

concentration of either G6P or NADP+ in the presence of saturating concentrations of the

other substrate (0.3 mM G6P or 0.20 mM NADP+). The kinetic data were analysed

graphically to determine the type of inhibition whereas the Ki value was determined from the

corresponding reciprocal plot.

Thermostability of the purified G6PD

For thermostability experiments, concentrated purified enzyme in 50 mM potassium

phosphate buffer, pH 7.5 containing 10 mM MgCl2 was incubated for 60 min at various

temperatures between 25 and 55 °C and percentage of residual activity was determined at

different time intervals.

Total protein determination

Total protein concentration was assayed by the method of Bradford [20]. A calibration curve

was established using BSA as a standard at a concentration of 0.5 mg/ml.

Purification of G6PD by immobilized metal affinity fast protein liquid chromatography

The supernatant containing recombinant G6PD was loaded onto a 1 ml HiTrap HP nickel-

nitrilotriacetic acid (Ni-NTA) column operated in an ÄktaPurifier system (column and Äkta

from GE healthcare). The FPLC system was set at a flow rate of 1 ml/min throughout the

operation. The column was washed thoroughly by using 10 column volumes of equilibration

buffer (50 mM potassium phosphate buffer pH 7.5 containing 300 mM NaCl, and 20 mM

imidazole). Recombinant G6PD was recovered using an elution buffer containing 50 mM

potassium phosphate buffer pH 7.5 containing 300 mM NaCl and 500 mM imidazole and

collected in 2 ml fractions. Fractions with high enzymatic activity were combined and

concentrated by ultrafiltration with Amicon Ultra-15 30K (Millipore) at 4000 × g for ∼1 h.

SDS-PAGE and western blotting analysis

The expression of recombinant G6PD and the progress of purification steps were monitored

by electrophoresis on 10% SDS–polyacrylamide gel according to Laemmli [21].

Recombinant fusion C. dromedarius G6PD protein expressed in E. coli BL21 (DE3) pLysS

was detected by western blotting using anti-human-G6PD polyclonal antibody produced in

rabbit (Sigma–Aldrich Life Science Cat. # HPA000247). Proteins were separated by

electrophoresis on 10% SDS–polyacrylamide gel and transferred to nitrocellulose membrane

according to Towbin et al. [22]. After electrophoretic transfer, membrane was blocked for 3 h

with 50 ml of 1x blocking buffer (Sigma–Aldrich Cat. # B6429). Membrane was then

incubated first with the primary anti-human-G6PD antibody (1:000 dilution) for 8 h then

washed for 30 min with 1x TBS containing 0.05% Tween-20. After a final wash with 1x TBS

buffer, membrane was incubated for 1h with anti-rabbit secondary alkaline phosphatase

labelled IgG antibody. Membrane was washed as described before and finally was developed

using NBT/BCIP substrates. Protein bands were photographed using Alpha Imager System

(Alpha Innotech. Version: 2.0.0.9).

Isoelectric focussing of recombinant G6PD

IEF polyacrylamide gel electrophoresis was performed on a Multiphor II system provided

with a MultiTemp III refrigerated bath circulator (Amersham Pharmacia Biotech, Uppsala,

Sweden). The pI of the purified G6PD was determined by analytical electrofocusing on 5%

thin-layer polyacrylamide flat gel containing ampholytes (pH 3.0–10.0). Broad pI Calibration

Kit, (Amersham Pharmacia Biotech) protein standards, with known isoelectric points, was

used as a reference. Isoelectric focusing was run at a constant power of 25 W for 1.5 h, with 1

M H3PO4 in the anode strip and 1 M NaOH in the cathode strip at 10 °C. Bands are

visualized by staining with Coomassie Brilliant Blue G-250.

Circular dichroism

Spectropolarimeteric measurements were made with Chirascan™-plus CD Spectrometer

(Applied Photophysics Ltd., Leatherhead, UK). To deconvolute Far-UV CD spectra, CDNN

software package (version 2.1) provided by Applied Photophysics Ltd., Leatherhead, UK was

used. The far-UV and near-UV measurements of G6PD were done on chirascan Plus

Spectropolarimeter equipped with peltier temperature controller at 20 oC. The CD instrument

was calibrated with (1S)-(+)-10-Camphorsulfonic acid. For far-UV measurements,

monochromator was set at 260 nm with 1 nm bandwidth. The CD spectra of G6PD (1.5 µM)

were collected between 180-260 nm using 1mm path length cuvette. Data sampling was made

using 0.5 current time per-point and 0.5 second current time per-point. In the near-UV

measurements, monochromator was set at 330 nm with 0.5 nm bandwidth. The near UV CD

spectra of G6PD (7.5 µM) were recorded in 10 mm path length cuvette at 1 second time per

points between 250-330 nm. To determine conformational change in secondary and tertiary

structure induced by NADP+ binding, 1.5 and 7.5 µM G6PD was incubated with 50 µM

NADP+ for far and near-UV CD, respectively. Measurements were made in identical

condition. In both far and near-UV CD, 4 repeats of measurements were averaged and air

and buffer controls were blanked from respective spectra. The secondary structure content

was calculated using CDNN software.

Results and discussion

Over expression and purification of C. dromedarius G6PD

Life of an organism like the Arabian camel is complicated and interplay between the

structural-functional integrity of biological system and the influence of various environmental

conditions. The projected increase in environmental pollution indicates that it is worthwhile

to study an animal models that competes under such conditions. The one-humped camel

appears to be the best model of a species that thrives in this type of Arabian Desert

environment. The camel developed unique adapted mechanisms that allow it to withstand

harsh desert environment such as direct exposure to high temperatures that may result in

oxidative stress and cellular damage [19, 23-25]. One of the most important housekeeping

genes is the G6PD which plays a significant role in maintaining the levels of NADPH for

fatty acid synthesis, generation of pentose sugars for nucleotide synthesis and elimination of

the generated toxic ROS resulted from different metabolic pathways and environmental

insults [4]. G6PD has been extensively studied in recent years in many different species but

the information about the Arabian camel G6PD is scarce [2, 27]. The elucidation of the

Arabian camel G6PD function and structure relies on the availability of highly purified

protein in sufficient quantities for detailed biochemical and biophysical characterization. To

this end, simple recombinant production system on pET28a (+) vector is highly valuable. In

an earlier study, we reported for the first time the isolation and characterization of the

Arabian camel C. dromedarius complete coding sequence of G6PD cDNA and its expression

in E. coli cells [2]. To complement what has been investigated in the previous study we

reported here over-expression and characterization of the recombinant G6PD from the

Arabian camel. At all steps of purification of the Arabian camel recombinant G6PD, glycerol

was added at a concentration of 10 % to preserve the enzyme in its active form by preventing

the aggregate formation [26]. Escherichia coli BL21(DE3) pLysS cells transformed with

pET28a (+) G6PD were 2 L of Terrific broth and high level of recombinant G6PD was

attained by adding 2.0 mM of the inducer IPTG for 18 hours at 37 °C (Fig. 1 A). The Arabian

camel recombinant G6PD was found to be immunoreacted with anti-human G6PD antibody

giving a band of 63.0 KDa corresponding to the molecular weight of the recombinant fusion

protein. No immunoreactivity was detected in the un-induced E.coli BL21 (DE3) pLysS cells

harbouring pET28a (+) g6pd (Fig. 1 B). The Arabian camel recombinant G6PD was purified

utilizing a rapid and efficient procedure on immobilized metal affinity fast protein liquid

chromatography. The elution profile was shown in Fig. 2. Recombinant G6PD was bound to

the nickel affinity matrix and the fractions containing G6PD were eluted by imidazole at a

concentration of 500 mM. Purification procedures were monitored on SDS-PAGE (Fig. 1C).

The purified enzyme was eluted as a single protein band of 63.0 KDa. The specific activity of

the purified G6PD was determined to be 2000 U/mg protein and 29.85 fold increase in the

purity was obtained. The overall yield was about 88.78% and the total yield of the purified

recombinant enzyme was found to be 140 mg per litre of TB medium (Table 1). The

isoelectric point of the enzyme was determined by chromatofocusing (Fig. 1D) as 6.4 and this

value is in agreement with the native Arabian camel G6PD isolated and purified from the

liver [27]. Lower isoelectric point value was reported for G6PD from bovine lens at 5.14

[16].

Effect of temperature, pH and thermal stability on G6PD

The purified recombinant G6PD was active at different temperatures ranging from 25 to 75

°C with maximal activity at 60 °C (Fig. 3A). The enzyme was active in the pH range 7-9 with

optimal pH at 7.5 in the assay buffer containing 10% glycerol (Fig. 3B). It was reported that

the camel liver native G6PD displayed optimum pH at 7.8 [27]. Similarly, the pH optimum

for human placenta G6PD was determined to be 7.8 [28], lamb kidney cortex was 7.8 [29]

and for sheep kidney cortex was 7.4 [30]. Thermal stability of the Arabian camel recombinant

G6PD was tested by determination of the residual enzyme activity percent after exposure of

the purified enzyme preparations at different temperatures. Result in Fig. 3C showed that the

recombinant G6PD retained 100% of the activity at 25 °C for 60 min. Moreover, the enzyme

retained 50.8% of its original activity after incubation at 37 °C for 60 min, on the other hand

exposure at 65 °C for 60 min only 4% of the original activity was retained (Fig. 3C).

Kinetics properties of the recombinant G6PD

The recombinant Arabian camel G6PD showed typical Michaelis-Menten kinetics for both

NADP+ and G6P. Km values were calculated from the secondary plots of the slopes and

intercepts plotted against their reciprocal concentration in the primary plot (Fig. 4 A-D). The

apparent Km values for the two substrates NADP+ and G6P were found to be 23.2 µM and

66.7 µM respectively, which is similar to that reported for G6PD from other sources [28-30].

The Km value for Arabian camel recombinant G6PD was found to be substantially higher

than that from other animal sources such as horse liver, rat liver, mouse liver and human

placenta, suggesting that the camel recombinant G6PD has lower affinity for G6P and higher

affinity towards NADP+. The analysis of double reciprocal plots obtained from the purified

recombinant G6PD by varying substrate concentrations were utilized in order to define a

reaction mechanism for the Arabian camel liver recombinant G6PD (Fig. 4). The intersecting

pattern of double plots with either G6P or NADP+ as variable substrates are coherent with a

sequential mechanism, in which both substrates must bind to the enzyme concurrently before

product formation. This suggest that, the binding of NADP+ slightly increases the binding of

G6P and the binding of G6P as the first substrate does not affect the binding of NADP+. It

was reported that, G6PD from some other sources such as fungi (Saccharomyces

carlsbergennis, Penicilin duponti) and mammals (rabbit erythrocytes, rat breast, bovine

adrenals), follow random sequential mechanisms [31]. Studies on recombinant human G6PD

suggested that the enzyme follows a random mechanism as well [28]. It was found that,

recombinant camel liver G6PD was inhibited by NADPH. Double reciprocal plot of the

initial velocity data revealed a competitive inhibition pattern with respect to NADP+ and non-

competitive with respect to G6P (Fig. 5 A and B). A plot of the slopes against the NADPH

concentrations gave a Ki of 38 µM. Competitive inhibition of G6PD from different sources

by NADPH with various values of Ki was reported reflecting the metabolic regulation of

G6PD activity by NADP+/NADPH ratio [31-32].

CD spectroscopy

Previously, the predicted 3D structure model of G6PD indicated dominance of α-helical

structure [2]. Analyses of CD spectrum of purified recombinant 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 (Fig. 6A) [2, 33]. In the far-UV region, the different secondary

structures give rise to characteristic CD spectra due to conformation of peptide bonds. The

far-UV CD spectra of the G6PD in the presence or absence of NADP+ were nearly identical

(Fig. 6B), which indicated that, NADP+ binding did not notable alter the secondary structures

of the G6PD. The content of the secondary structure in G6PD in the presence or absence of

NADP+ (Table 2) was calculated from ultraviolet region of the CD spectrum by CDNN CD

software [34]. It was observed that, the binding of NADP+ to the G6PD induced insignificant

changes in the secondary structure. Changes in near-UV spectra provide evidence of tertiary

structure as well as dynamics. Tryptophan and tyrosine shows strong signal between 270 to

305 nm. Our results showed that spectra overlap between (274 to 330 nm) in the presence or

absence of NADP+, indicating no environmental changes around tryptophan and tyrosine

residues. However, in the NADP+ bound state G6PD CD spectra exhibited an additional peak

around 268 nm. The CD signal of phenylalanine arises due to 1Lb electronic transition with

low intensity at 262 and 268 nm. The occurrence of peak at 268 nm in NADP bound state

indicates conformational change in G6PD, leading to localization of phenylalanine in highly

specific and well-packed environment [35].

In conclusion, G6PD plays an important role in maintaining the level of NADPH and in the

production of pentose sugar for nucleotide biosynthesis. The stability of the G6PD can be

affected by several factors such as pH, temperature and the requirement for coenzymes and

cofactors. The present study was conducted with the aim of overexpression in E. coli of the

Arabian camel G6PD, purification and characterization of the affinity purified enzyme. The

data provided here will allow the further study of the Arabian camel G6PD gene such as

analysis of the 5’ regulatory region of camel G6PD gene as a tool to examine the mechanism

of lipogenesis in this unique important animal.

Acknowledgement

The authors extend their appreciation to the Deanship of Scientific Research at King Saud

University for funding the work through the research group project number RGP-VPP-173.

Conflict of interest

I declare that there is no conflict of interest for this article and there is no financial

employment, consultancies, honoraria, stock ownership or options, expert testimony, grants

or patents received or pending, royalties related to this manuscript. Moreover, I declare that

this work did not published elsewhere; did not simultaneously submitted for publication

elsewhere and all authors agree to the submission.

Author contributions

All authors participated in the design, interpretation of the studies and analysis of the data and

review of the manuscript.

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Figure Legends

Fig. 1: Panel A, Sodium dodecyl sulphate polyacrylamide gel (12%) electrophoresis of un-induced

E. coli BL21(DE3)pLysZ pET-28a(+) carrying C. dromedarius G6PD cDNA (Lanes 2-4) and 2 mM

IPTG induced culture (Lanes 5-9). Panel B, immunoblotting analysis of un-induced (Lanes 2-4) and

induced (Lanes 5-9) cultures of C. dromedarius recombinant G6PD protein with anti-human G6PD

antibody. Lane 1 represents Precision Plus Prestained Protein Standard from BioRad, Cat # 161-0373.

Panel C, SDS-PAGE (12%) for nickel affinity column washed sample (Lane 3) and 0.5 M imidazole

eluted enzyme (Lane 2). Lanes 1 and 4 represent protein standard markers. Panel D, non-denaturing

isoelectric focusing protein gel for purified recombinant C. dromedarius G6PD (Lanes 2).

Lane 1 represents pI markers peptides.

Fig. 2: Purification pattern of the Arabian camel recombinant G6PD on immobilized nickel

affinity fast protein liquid chromatography. Elution was monitored at 280 nm (blue line).

Fig. 3: Temperatures (A), pH optimum (B) and thermal stability at ♦ 25 °C, ■ 37 °C, ▲ 45 °C

and ■ 55 °C (C) of the purified recombinant Arabian camel G6PD.

Fig. 4: Primary double reciprocal plots of 1/v against 1/[S] at various concentrations of G6P

(A), ● 20µM, ■ 30µM, ▲ 40µM, ▼ 60µM, ♦ 100 µM and □ 300µM. (B) At various

concentrations of NADP+, ● 10µM, ■ 20µM, ▼ 40µM, ▲ 60µM, ○ 100µM and □ 200µM.

(C) Secondary plots of slopes of primary plots vs. 1/ [NADP+]. (D) Secondary plots of

intercepts of primary plots vs. 1/ [G6P].

Fig. 5: Inhibition studies of recombinant camel liver G6PD by the product inhibitor NADPH

with varied concentrations of G6P (A), ● 0µM, ♦ 10µM, □ 20µM, ∇ 40µM and ▼ 80µM. (B)

At varied concentrations of NADP+ (B).

Fig. 6: Circular Dichroism spectra of camel G6PD. (A) Far-UV CD in the absence (solid

line) and presence of NADP (dotted line). Protein concentration was 1.5 mM in 10 mM

phosphate buffer, pH 7.4 and path length 1 mm. (B) Near-UV CD in the absence (solid line)

and presence of NADP (dotted line). Protein concentration was 7.5 mM in 10 mM phosphate

buffer, pH 7.4 and path length 10 mm.

Fig. 1:

KDa 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

50

150

37

25

20

250

100

75

50

150

37

25

20

250

100

75

KDa 1 2 3 4 pI Markers 1 2

8.6

8.48.1

7.3

6.5

5.8

5.2

4.5

A B C D

Fig. 2:

Table 1: Purification summary of the Arabian camel recombinant G6PD

Purification

step

Total volume

(mL)

Activity (EU/mL)

Total activity

(EU/mL)

Protein

(mg/mL)

Specific activity

(EU/mg)

Yield (%)

Purification

fold

Cell free homogenate

Nickel affinity purified G6PD

15

18

378.46

280

5676.9

5040

5.65

0.14

66.98

2000

100

88.78

1.0

29.85

0

100

200

300

400

500

600

700

800

20 30 40 50 60 70 80

Act

ivit

y (U

/ml)

Temperature (°C)

0

50

100

150

200

250

300

350

3 5 7 9 11

Act

ivit

y (U

/ml)

pH values

0

20

40

60

80

100

120

0 10 20 30 40 50 60

Res

idua

l A

ctiv

ity

(%)

Exposure Time (min)

A B

C

Fig. 3

-0.04 -0.01 0.02 0.05 0.08 0.11

0.05

0.10

0.15

0.20

0.25

1/[NADP] µµµµM

1/V

( µµ µµm

ol m

in-1

mg

-1)

0.00 0.02 0.04

0.01

0.02

0.03

0.04

66.7

1[G6P] µµµµM-1

Inte

rce

pt

-0.02 0.00 0.02 0.04 0.06

0.06

0.12

0.18

0.24

0.30

1/[G6P] µµµµM

1/v

(µµ µµ

mo

l m

in-1

mg

-1)

0.00 0.04 0.08 0.12

1

2

3

23.2

1/[NADP+] µµµµM

Slo

pe

Fig. 4 A, B, C and D:

A

B

C

D

-0.04 0.00 0.04 0.08 0.12 0.16 0.20

0.1

0.2

0.3

0.4

1/[NADP] µµµµM

1/V

(µ

ml/m

in/m

g)

0 20 40 60 80 1000.0

0.4

0.8

1.2

1.6

2.0

37.9

[NADPH] (µM)

Slo

pe

-0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5

0.1

0.2

0.3

[g6p] µµµµM

1/V

(µ

ml/m

in/m

g)

0 20 40 60 80 1000.00

0.02

0.04

0.06

0.08

55.5

[NADPH] (µM)

Inte

rce

pt

Fig. 5 A and B:

A

B

Table 2: Relative contents (%) of particular secondary structures of G6PD, calculated from185-260 nm CD spectra

Secondary Structure G6PD G6PD + NADP+

Helix

Anti-parallel β-sheet

Parallel β-sheet

β-turn

Random coil

30.2%

11.6%

9.0%

17.6%

33.3%

30.2%

11.7%

9.0%

17.7%

33.2%

Fig. 6 A and B:

Wavelength (nm)

260 280 300 320

CD

(md

eg

)

-8

-6

-4

-2

0

2

Wavelength (nm)

180 200 220 240 260

CD

(md

eg

)

-6

-3

0

3

6

9

A B

Highlight

♦ Camelus dromedarius recombinant G6PD was heterologously overexpressed in Eschericia

coli BL21 (DE3) pLysS and purified by immobilized metal affinity fast protein liquid

chromatography (FPLC) in a single step.

♦ Recombinant G6PD was found to be immunoreacted with anti-human G6PD antibody

giving a band of 63.0 KDa corresponding to the molecular weight of the recombinant fusion

protein.

♦ The pI was found to 6.4 and this value is in agreement with the native Arabian camel G6PD

isolated and purified from the liver.

♦ 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 of the G6PD.