Abstract
A sec-type protein secretion is an alternative approach to recombinant protein production, where protein expression and purification are simultaneously achieved. In this study, uricase (urate oxidase, EC 1.7.3.3) was fused with a sec-type Epr signal peptide to achieve the extra-cellular uricase production in Escherichia coli MG1655. In shake-flask experiments, the enzyme activity and the specific activity in the extra-cellular fraction reached 0.73 ± 0.05 U/mL and 3.17 ± 0.22 U/mg-protein, respectively. When a 3-L bioreactor was used, the extra-cellular fraction exhibited the enzyme activity of 0.74 ± 0.00 U/mL and the specific activity of 2.75 ± 0.35 U/mg-protein. It was estimated that 89 % and 72 % of total enzyme activity as produced were found in the extra-cellular fraction in the shake flask and the 3-L bioreactor, respectively. The uricase secretion in E. coli was further supported by SDS-PAGE. In contrast, no uricase activity was found when Bacillus subtilis 168 was used as the host. This study demonstrates the efficacy of protein secretion using the Epr signal peptide in E. coli MG1655. Finally, this study presents that acidic pH results from the acetate production could be detrimental to extra-cellular uricase production.
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