title: 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.
authors.affiliation: Microbiology Department, Faculty of Pharmacy, Mansoura University, Egypt.
authors.forename: Mona I
authors.initials: MI
authors.lastname: Shaaban
authors.affiliation: Microbiology Department, Faculty of Pharmacy, Mansoura University, Egypt.
authors.forename: Eman
authors.initials: E
authors.lastname: Abdelmegeed
authors.affiliation: Microbiology Department, Faculty of Pharmacy, Mansoura University, Egypt.|Department of Infection, Immunity and Inflammation, University of Leicester, LE1 9HN, UK.
authors.forename: Youssif M
authors.initials: YM
authors.lastname: Ali
mesh_terms: D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014503:Urate Oxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D014503:Urate Oxidase