title: Cloning and expression of a urate oxidase and creatinine hydrolase fusion gene in Escherichia coli.
abstract: OBJECTIVE
  To construct a plasmid containing a urate oxidase and creatinine hydrolase fusion gene and transform the plasmid into Escherichia coli to decompose uric acid and creatinine.
  
  
  METHODS
  According to the GenBank data for the urate oxidase gene, specific primers were designed to amplify and remove the stop codon for the urate oxidase gene. The gene was then ligated into the plasmid pMG36e to construct pMG36e-U. Then, using the GenBank database for the creatinine hydrolase gene, primers were designed to amplify the creatinine hydrolase gene. This gene was ligated into pMG36e-U to form pMG36e-U/C. Next, this construct was transformed into E. coli, which was confirmed by screening the recombinant E. coli and sodium dodecylsulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The engineered bacteria were cultured with a specific concentration of creatinine and uric acid for 24 h. Then, the concentrations of creatinine and uric acid in the culture fluid were measured.
  
  
  RESULTS
  The recombinant gene fragment was approximately 1.68 kb, and it contained the urate oxidase and creatinine hydrolase genes. The transformed E. coli expressed creatinine hydrolase and uric acid oxidase. The creatinine decomposition rate increased by 43.5%, and the uric acid decomposition rate increased by 42.32%.
  
  
  CONCLUSIONS
  The constructed recombinant plasmid containing a fusion gene of creatinine hydrolase and uric acid oxidase was transformed into E. coli, and the enzymatic activities were expressed.
authors.affiliation: Division of Nephrology, The Second Xiangya Hospital, Research Institute of Nephrology, Central South University, Changsha, PR China.
authors.forename: Xin
authors.initials: X
authors.lastname: Cheng
authors.forename: Fang
authors.initials: F
authors.lastname: Liu
authors.forename: Yanxin
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yunsheng
authors.initials: Y
authors.lastname: Jiang
mesh_terms: D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D012016:Reference Values; D012680:Sensitivity and Specificity; D014503:Urate Oxidase / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014503:Urate Oxidase; D000581:Amidohydrolases; C019946:creatininase