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 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.

Combinatorial approach for screening and assessment of multiple therapeutic enzymes from marine isolatePseudomonas aeruginosaAR01

“…Several reports are available on uricase from Pseudomonas aeruginosa with an enzyme activity of 17.77 U per mL and 13.42 U per mL for P. aeruginosa 5Y2 and P. aeruginosa Ph3 respectively. 17,18 Successful cloning and high-level expression of native uricase gene from P. aeruginosa Ps43 were reported by Shaaban et al 22 Some strains exhibit signicantly higher enzyme activity of 25.88 U per mL at 12 h of incubation at a uric acid concentration 0.3% (w/v). 19 The enzyme activity declined when the incubation was further extended as well as an increase in the substrate concentration.…”

“…For example, a Pseudomonas strain was recently isolated that can utilize TNT (2,4,6trinitrotoluene) as a sole nitrogen source, producing toluene, aminotoluene and nitrotoluenes as end products. 8 A wide variety of extracellular enzymes such as asparaginase, [9][10][11][12][13][14] glutaminase, 11,15 uricase [16][17][18][19][20][21][22] and collagenase [23][24][25][26] were reported in Pseudomonas aeruginosa.…”

Efficient purification of a recombinant tag-free thermostable Kluyveromyces marxianus uricase by pH-induced self-cleavage of intein and expression in Escherichia coli

“…Furthermore, uricase with a 4-aminoantipyrine peroxidase system is extensively employed as a reagent in the clinical analysis of uric acid in serum (Liu et al 1994). So far, many uricases from various microbes, including Candida utilis (Koyama et al 1996), Bacillus subtilis (Pfrimer et al 2010), Aspergillus flavus (Legoux et al 1992), and Pseudomonas aeruginosa (Shaaban et al 2015), have been recombinantly expressed. Rasburicase mentioned above is the recombinant uricase cloned from A. flavus and expressed in Saccharomyces cerevisiae (Pui et al 2001).…”

Expression and purification of human IL-2 protein from Escherichia coli

“…The protein concentration was calculated from the standard curve of bovine serum albumin (positive control) after measurement of the absorbance at 595 nm using ELx808™ Absorbance Microplate Reader (Biotek Instruments Inc.,Winooski, VT) (Shaaban et al, 2015). The buffer containing the purified IL-2 protein (100 mM Tris-HCl, pH 7) was used as blank (negative control).…”

“…Western blot analysis of the expressed IL-2 proteins was performed using diluted (1:1000) 6-Histidine Epitope Tag Antibody [HRP] (Novus Biologicals, USA) as previously mentioned in El- Mowafy et al (2013), except that the His-tagged protein bands were visualized by incubation of the membrane with tetramethylbenzidine (TMB) substrate solution (Sigma Aldrich, USA) at room temperature for few minutes before capturing of photos (Shaaban et al, 2015).…”