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%. Conclusion: 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.

• genetically engineered bacteria
• urate oxidase
• creatinine hydrolase
• fusion gene