June 2015⎪Vol. 25⎪No. 6

J. Microbiol. Biotechnol. (2015), 25(6), 887–892http://dx.doi.org/10.4014/jmb.1410.10041 Research Article jmbReview

Cloning, Expression, and Purification of Recombinant Uricase Enzymefrom Pseudomonas aeruginosa Ps43 Using Escherichia coliMona I. Shaaban1, Eman Abdelmegeed1, and Youssif M. Ali1,2*

1Microbiology Department, Faculty of Pharmacy, Mansoura University, Egypt2Department of Infection, Immunity and Inflammation, University of Leicester, LE1 9HN, UK

Introduction

Uric acid is the metabolic end product of purine metabolism

in humans and higher vertebrates [27]. Accumulation of

uric acid in blood is a predisposition factor for gout disease

and acute or chronic renal nephropathy [13-27]. Moreover,

precipitation of urate crystals leads to formation of the

renal calculi, intestinal necrosis, and skin calcification [5].

Uricase is an essential enzyme that catalyzes the oxidative

degradation of uric acid in the presence of oxygen to

allantoin, which is a more soluble form than uric acid and

can be easily excreted by the kidney [29]. Uricase enzyme is

commonly present in bacteria and fungi, but higher

organisms (such as humans) are devoid of uricase owing to

mutations in the uricase gene that introduces a premature

stop codon that terminates the translation process and

inhibits enzyme production [24]. Uricase has been used as

a therapeutic agent to reduce toxic urate accumulation and

hence as treatment of gout disorders and nephropathy [14].

Recombinant uricase enzyme was successfully used in

the treatment of hyperuricemia, with low incidence of

hypersensitivity, and successfully used for treatment of

tophaceous gout [26, 32]. It is also widely used as a reagent

in clinical diagnostic kits for enzymatic determination of

uric acid by coupling with the 4-amino antipyrine-

peroxidase system [9].

Purification of native uricase enzyme has been reported

from various microorganisms, including fungi, yeast, and

bacteria. Some microbial sources of urate oxidase are

available such as Nocardia farcinica [17], Proteus vulgaris and

Microbacterium species [36], and Bacillus subtilis [25].

Recombinant uricase from Candida utilis was successfully

expressed in Hansenula polymorpha under the control of the

methanol oxidase promoter using Saccharomyces cerevisiae

alpha-factor signal peptide as the secretory sequence [7].

Purified uricase from Pseudomonas has been studied for

diagnostic purposes since it exhibits high activity and

thermostability in a wide range of temperatures [28].

However, to our knowledge, recombinant expression of

uricase from P. aeruginosa was not reported. This prompted

Received: October 20, 2014

Revised: January 8, 2015

Accepted: January 12, 2015

First published online

January 15, 2015

*Corresponding author

Phone: +44 116 252 3016;

Fax: +44 116 2525030;

E-mail: ya64@alumni.le.ac.uk;

sarafawzy2002@yahoo.com

pISSN 1017-7825, eISSN 1738-8872

Copyright© 2015 by

The Korean Society for Microbiology

and Biotechnology

Uricase is an important microbial enzyme that can be used in the clinical treatment of gout,

hyperuricemia, and tumor lysis syndrome. A total of 127 clinical isolates of Pseudomonas

aeruginosa were tested for uricase production. A Pseudomonas strain named Ps43 showed the

highest level of native uricase enzyme expression. The open reading frame of the uricase

enzyme was amplified from Ps43 and cloned into the expression vector pRSET-B. Uricase was

expressed using E. coli BL21 (DE3). The ORF was sequenced and assigned GenBank Accession

No. KJ718888. The nucleotide sequence analysis was identical to the coding sequence of

uricase gene puuD of P. aeruginosa PAO1. We report the successful expression of P. aeruginosa

uricase in Escherichia coli. E. coli showed an induced protein with a molecular mass of about

58 kDa that was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and

Western blotting. We also established efficient protein purification using the Ni-Sepharose

column with activity of the purified enzyme of 2.16 IU and a 2-fold increase in the specific

activity of the pure enzyme compared with the crude enzyme.

Keywords: Uricase, expression, recombinant, P. aeruginosa, western blot

888 Shaaban et al.

J. Microbiol. Biotechnol.

us to test for expression of P. aeruginosa uricase in E. coli,

which showed higher activity than other uricases isolated

from different microorganisms.

Materials and Methods

Microorganisms and Media Composition

Escherichia coli DH5α (Invitrogen, Carlsbad, CA, USA) was used

as the host for plasmid clones. E. coli BL21 (DE3) pLysS cells (F-,

ompT hsdSB (rB- mB-) gal dcm λ (DE3) pLysS) (Invitrogen, Carlsbad,

CA, USA) was used for protein expression. Bacterial strains were

propagated using LB medium (Luria-Bertani) containing 1% (w/v)

bactotryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl; and for LB

agar 2%, (w/v) agar was added, pH 7-7.5. Ampicillin (100 µg/ml)

and chloramphenicol (35 µg/ml) were added as required. Plasmid

pRSET-B (Cat. No. V351-20 Invitrogen, Carlsbad, CA, USA) was

used for uricase expression under the control of the T7-promoter

induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) (Sigma

Aldrich, USA).

Collection, Purification, and Identification of P. aeruginosa Isolates

One hundred twenty-seven clinical isolates of P. aeruginosa

were obtained from specimens collected from Mansoura University

hospitals, Dakhlia, Egypt. The clinical isolates were obtained from

various sources, including 41 isolates from urine, 26 isolates from

wound infection, 24 isolates from sputum, 11 isolates from burn, 9

isolates from blood, 9 isolates from endotracheal tube, 6 isolates

from throat, and one isolate from ear. The isolates were purified

and identified biochemically as P. aeruginosa according to

laboratory biochemical standards [10].

Screening of Uricase

Primary screening of uricase from different clinical isolates was

performed by inoculating the test organisms onto agar plates with

uric acid medium (uric acid 0.3% (w/v), beef extract 0.3% (w/v),

peptone 0.5% (w/v), NaCl 0.2% (w/v), and agar 2.3% (w/v) at

pH 7.5) incubation at 37ºC. The appearance of clear zones around

the Pseudomonas colonies is an indication for uricase enzyme

production [20].

Quantitative Estimation of Uricase Enzyme Level

Estimation of the uricase enzyme level was done by the uricase

plate assay method according to the procedure described by

Lehejckova et al. [20]. A single colony of the purified Pseudomonas

strains was grown for 24 h in LB medium. The OD600 nm of the

Pseudomonas cultures was adjusted to 1 using sterile LB medium.

Pseudomonas PAO1 was used as the standard strain for the assay.

The Pseudomonas pellet was separated by centrifugation at 8,000 ×g

for 5 min and the supernatant was tested for uricase production.

Cups (10 mm diameter) were made in uric acid agar media using

a sterile cork pourer and 100 µl of Pseudomonas supernatant was

added to each well and incubated at 37ºC for 24 h. Uricase

production was identified as a clear zone around the well due to

degradation of uric acid. The diameter of uricase clearance zone

was measured in millimeter unit by a graduated caliper. P. aeruginosa

(isolate Ps43) showed the highest level of uricase activity and was

chosen for recombinant engineering and enzyme expression.

Plasmid Construction

Chromosomal DNA of P. aeruginosa Ps43 was extracted using a

genomic DNA extraction kit (Qiagen Inc., USA) according to the

manufacturer instructions. The open reading frame of uricase

enzyme was PCR amplified using the following pair of primers;

Uricase_F 5’-ATTGGATCCGGACCCTCTTCCCGGAGAAG-3’ and

Uricase_R 5’-TATCCATGGATGAGTTCCTCGAGCTGGAAGTG-

3’. The primer pair introduced restriction sites for BamHI and NcoI

respectively for subsequent cloning into the bacterial expression

vector pRSET-B in frame with and downstream of nucleotides

encoding a 6-histidine tag sequence.

Expression of Uricase Enzyme

The resulting expression construct was named as pU3. pU3 was

transformed into E. coli BL-21 (DE3) pLysS and positive transformed

cells were selected on LB agar plates with 100 µg/ml ampicillin

and 35 µg/ml chloramphenicol. E. coli BL21 (DE3) transformed

with pU3 was grown in 500 ml of LB medium at 30ºC with

shaking until OD 600 nm of 0.4-0.5 was reached and 5 ml of

culture supernatant was removed. Uricase expression was induced

using 1 mM IPTG for 24 h. Samples (5 ml) were taken each hour

for the first 5 h and then after 24 h. The cell pellet was harvested by

centrifugation at 8,000 ×g for 30 min at 4ºC and resuspended in

PBS (phosphate buffer saline) (pH 8.0). The bacterial cell pellet

was sonicated at 4°C using a Soniprep 150 sonicator (TM-182;

UK). Cell debris was centrifuged at 8,000 ×g for 20 min at 4ºC. The

supernatant containing the soluble recombinant protein was

purified using a Ni+2 Sepharose column (HisGravi Trap column;

GE Healthcare, Sweden) according to the manufacturer instructions.

The column was first washed with 10 ml of distilled H2O and then

equilibrated with 10 ml of PBS. The cell supernatant was diluted

with an equal volume of 2× phosphate buffer, pH 7.4 (contained

100 mM NaCl and 5 mM imidazole), and loaded on a HisGravi

Trap column. The column was washed with 20 ml of phosphate

buffer, pH 7.4, containing 100 mM NaCl and 25 mM imidazole

(wash buffer). The recombinant protein was eluted using elution

buffer (PBS containing 300 mM imidazole). Protein fractions were

analyzed by SDS-PAGE, and stained with Coomassie Brilliant

Blue [19]. Fractions containing recombinant uricase protein were

pooled and washed several times with PBS to remove excess

imidazole concentration using Amicon Ultra 3K Centrifugal Filter

Devices (Millipore, USA).

Western Blot Analysis

Western blot analysis of the expressed protein was performed

using diluted (1:4,000) anti-histidine-tag antibodies (Sigma Aldrich,

Expression and Purification of P. aeruginosa Uricase 889

June 2015⎪Vol. 25⎪No. 6

USA). Then the membrane was visualized by incubation with

tetramethylbenzidine (TMB) substrate solution at room temperature

(Sigma Aldrich, USA).

Determination of Protein Content

The protein concentration of the purified uricase was measured

using a Bradford protein assay kit (Fermentas, USA) according to

the manufacturer’s instructions. The protein content was calculated

from the standard curve [4].

The GenBank accession number for the sequence reported in

this paper is KJ718888.

Results

Primary Screening and Selection of Highly Producing

Uricase Strains

A total of 127 P. aeruginosa isolates were subjected to

primary screening for uricase production. Almost all

Pseudomonas isolates were positive for uricase production

and produced a zone of clearance on uricase assay media;

however 10 isolates showed no uricase activity.

Quantitative estimation of the uricase production was

performed by the uricase plate assay method. The enzyme

activity was calculated using a standard curve, where the

diameter of clearance zone was directly proportional to the

logarithmic concentration (Fig. 1A), and the uricase activity

of P. aeruginosa isolates from various clinical sources is

represented in Fig. 1B. P. aeruginosa No. Ps43 (from urine)

showed the highest level for uricase production (2.24 IU).

P. aeruginosa isolate (Ps43) was selected as a template for

amplification of the urate oxidase open reading frame

(ORF) and engineering of the recombinant vector.

Fig. 1. Screening of uricase activity using plate assay method.

(A) Standard curve for uricase assay. (B) Production of uricase enzyme by P. aeruginosa clinical isolates from various clinical sources; Ps127 (ear),

Ps43 (urine), Ps60 (sputum), Ps25 (blood), Ps116 (endotracheal tube), and Ps105 (burn).

Fig. 2. Amplification of uricase open reading frame and confirmation of the clones.

(A) Agarose gel electrophoresis of uricase open reading frame. Lane M: 1 kbp DNA size marker; and lane 2: the amplicon of uricase ORF

(1,458 bp). (B) Agaraose gel electrophoreses of EcoRI digestion of pU3 recombinant clone. Lane M: 1 kbp DNA size marker, and lane 2: pU3

plasmids digested with EcoRI with two bands at 1,299 and 3,086 bp. (C) Diagnostic figure of pRSET-B vector and pU3 plasmid with insert ORF of

uricase enzyme.

890 Shaaban et al.

J. Microbiol. Biotechnol.

Amplification and Cloning of Uricase Enzyme ORF

The ORF of uricase enzyme (1,458 bp) was amplified

from the genomic DNA of Ps43 (Fig. 2A) and cloned into

expression vector pRSET-B. The positive clones were

selected into LB/amp agar. Recombinant plasmids were

digested with EcoRI, and clones containing the ORF of

uricase enzyme produced two bands at 1,299 and 3,086 bp

(Figs. 2B and 2C). The expression construct pU3 was also

digested with BamHI/NcoI to release the inserted gene, and

we got two bands at 1,458 and 2,900 bp representing

pRSET-B vector and the insert ORF of uricase, respectively.

Sequence Analysis of Recombinant Uricase ORF

The ORF of the recombinant clone pU3 was sequenced

and the nucleotide sequence was submitted to GenBank

and denoted Accession No. KJ718888. The nucleotide

sequence analysis was identical to the coding sequence of

the GenBank urate oxidase gene puuD of P. aeruginosa

PAO1 (Accession No. AE004091.2).

Expression and Purification of Recombinant Uricase Enzyme

The expression of uricase pU3 by E. coli BL21 was induced

with 1 mM IPTG. Maximum protein expression was reported

5 h post induction. The recombinant uricase enzyme

showed a high degree of purity following purification by

Ni+2 affinity chromatography. The recombinant uricase band

was detected as expected at approximately 58 kDa (Fig. 3A).

The identity of the protein was further confirmed by

western blot analysis using anti-histidine tag monoclonal

antibody (Sigma Aldrich, USA), where a clear band

corresponding to the molecular mass of the recombinant

uricase (58 kDa) was detected (Fig. 3B).

Determination of Uricase Enzyme Activity

The activity of purified uricase enzyme reached 2.16 IU.

It was almost similar to the activity of crude uricase enzyme

isolated from the original Ps43 Pseudomonas strain (2.24 IU).

However, the protein content of the crude enzyme and

purified uricase enzyme were 0.311 and 0.144 mg/ml,

respectively. Hence, the specific protein content of the

crude extract produced by Ps43 was 7.5. On the other hand,

the specific activity of purified enzyme was 15, with a 2-

fold increase in pure uricase specific activity compared

with the crude enzyme (Table 1). Moreover, the purified

enzyme retained 72% of enzymatic activity as compared

with the total recombinant protein (Table 1).

Discussion

Great interest has been directed toward the expression of

enzymes from bacterial sources for industrial, analytical,

and medical purposes. Uricase enzyme catalyzes the

oxidation of uric acid, a final product of purine catabolism,

to allantoin, which is more soluble and easily excreted than

uric acid [8]. High concentrations of uric acid in blood were

reported as a main reason for the development of gout

disease. For that reason, uricase is reported as a valuable

tool for therapeutic purposes [9].

Many microbial sources of uricase enzyme are available

but large-scale production is limited by the low expression

levels, lower stability, and difficulties in purification for

commercial implications [25]. To overcome these difficulties,

uricase enzyme has been expressed in heterologous

organisms [16]. Uricase enzyme has been reported to be

expressed from different microbes, including Candida utilis

[7], B. subtilis [25], and Aspergillus flavus [12, 21, 35].

A few studies reported the production of uricase from

Fig. 3. Expression of uricase enzyme using E. coli BL21 (DE3).

(A) SDS-PAGE of total and purified recombinant uricase stained with

Coomassie stain under reducing conditions (lane 1: purified protein;

and lane 2: total recombinant protein). (B) Western blot detection of

recombinant uricase using anti-histidine tag monoclonal antibody

(lanes 1 and 3: purified protein, and lane 2: total recombinant protein).

Table 1. Activities of crude and purified recombinant uricases.

Strain CompositionUricase enzyme activity

(IU)

Specific enzyme activity

(IU/mg protein )

Pseudomonas aeruginosa (Ps43) Crude extract 2.24 7.2

E. coli BL21 (DE3)/pU3 Total recombinant uricase enzyme 3 9

Purified uricase enzyme with a Ni+2 Sepharose column 2.16 15

Expression and Purification of P. aeruginosa Uricase 891

June 2015⎪Vol. 25⎪No. 6

P. aeruginosa strains with limited concentrations [28]. In our

study, 127 clinical isolates of P. aeruginosa were tested for

uricase production. Pseudomonas strains producing uricase

were identified by utilizing uric acid in the agar plate and

producing a clear zone of soluble allantoin (Fig. 1). Ninety-

three percentage of the isolates produced uricase, suggesting

its wide distribution among P. aeruginosa isolates. Pseudomonas

isolate Ps43 from urine has the highest levels of uricase

production (2.24 IU). In addition, Pseudomonas isolate Ps25

from blood showed high uricase yield (2.08 ± .01 IU).

In the following study, the expression of recombinant

uricase enzyme from Ps43 isolate in the expression vector

pRSET-B was performed in E. coli BL21 (DE3). However,

the ORF sequence was identical to that of uricase puuD of

P. aeruginosa PAO1 as the NCBI assigned AE004091.2.

Hence, the phenotypic screening of a high uricase-

producing strain was not the appropriate indication of the

genetic difference between the screened isolates. This

illustrated that the variability in uricase production among

the isolates was not related to genetic variations but due to

the metabolic versatility among Pseudomonas isolates [22].

P. aeruginosa isolates can adapt growth under various

conditions. Some studies showed that the presence of the

uric acid in the medium could induce uricase production

from A. niger [1, 2]. Moreover, the study of Essam et al. [11]

showed increased production of uricase from Proteus

vulgaris 1753 and B-317-C cultivated in the presence of uric

acid. P. aeruginosa isolates with uricase activity were

isolated from poultry wastes [3]. In our study, P. aeruginosa

Ps43 with the highest uricase level was isolated from urine

sample; the presence of high uric acid in the sample could

be a predisposing factor for high uricase activity. Previous

studies also indicated that the clinical sources markedly

affect the levels of elaborate exoproducts of P. aeruginosa

[33]. A condition such as mucoid phenotype of P. aeruginosa

was associated with cystic fibrosis patients [15]. High

elastase [34] and protease [18] were observed among

P. aeruginosa isolates from wound and sputum.

Interestingly, pU3 was successfully expressed in E. coli

BL21 (DE3) with soluble uricase enzyme activity of 3 IU.

Our purified enzyme also showed a 2-fold increase in its

specific activity (15 IU/mg) compared with the crude

uricase of Ps43 (7.2 IU/mg) (Table 1). At the same instance,

the uricase enzyme yield by a recombinant Hansenula

polymorpha strain harboring the Candida utilis uricase gene

was 2.6 IU/ml obtained by shaking flask technique [7].

However, the urate oxidase of Candida utilis expressed by

the recombinant Lactobacillus exhibited activity of 0.33 IU/

ml [6], which is much lower than our uricase yield of

2.16 IU/ml. Moreover, the maximum urate oxidase activity

of Apergillus flavus was 0.03974 IU/ml [31]. Recently,

Aspergillus flavus urate oxidase gene was cloned in the

pPICZαA expression vector and expressed in P. pastoris.

The amount of expressed urate oxidase (0.43 U/ml) in P.

pastoris was about 24.2% of total supernatant proteins [12].

Uricase enzyme was previously purified from P. aeruginosa

using the 70% ammonium sulfate precipitation method [28].

However, it is a partial purification method and it requires

further purification processing (dialysis and gel filtration

chromatography) for removing high salt concentrations. In

the following study, the presence of the N-terminal 6× His

tag of pU3 allowed a simple one-step uricase purification

using Ni+2 Sepharose column and retained 72% of its

enzymatic activity (Table 1), which was more that reported

for commercial Rasburicase (61%) [30]. Purification of

recombinant Bacillus uricase using magnetic beads and

nonspecific protease retained 67% of activity [23]. In

addition, purification of uricase from B. subtilis using a Ni-

NTA column was reported and the purified enzyme

retained 58% of the total recombinant enzyme [22].

The molecular mass of purified recombinant uricase as

determined by SDS-PAGE was estimated to be 58 kDa. It is

consistent with the theoretical molecular mass of the 486

amino acid protein deduced from its gene sequence and

linked to the histidine tag protein.

In conclusion, we reported the successful expression

and purification of a recombinant uricase enzyme from

P. aeruginosa in E. coli with production of the enzyme in a

soluble and active form.

Acknowledgments

This study was performed at the Laboratory of

Microbiology, Faculty of Pharmacy, Mansoura University,

Egypt. The authors have no financial conflict of interest.

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