Cloning, Expression, and Purification of Recombinant Uricase Enzyme from Pseudomonas aeruginosa Ps43 Using Escherichia coli

Abstract

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 puuDof 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.

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 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 a ording 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 37℃. 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 37℃ 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/mlampicillin and 35 µg/mlchloramphenicol. E. coli BL21 (DE3) transformed with pU3 was grown in 500 ml of LB medium at 30℃ with shaking until OD600 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 4℃ 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 4℃. 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, 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).

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.

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 3,086bp. (C) Diagnostic figure of pRSET-B vector and pU3 plasmid with insert ORF of uricase enzyme.

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).

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).

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).

Table 1.Activities of crude and purified recombinant uricases.

 

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 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). Ninetythree 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 uricaseproducing 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.