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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: [email protected];
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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