Development of Recombinant Escherichia coli expressing oxc and frc Gene of Oxalobacter formigenes / 대한비뇨기과학회지

ABSTRACT

PURPOSE: Oxalobacter formigenes (O. formigenes) is an obligate anaerobe, which may be important in the prevention of stone formation. O. formigenes degrades oxalates using oxalyl-CoA decarboxylase and formyl- CoA transferase encoded by the oxc and frc genes, respectively. Attempts were made to develop recombinant Escherichia coli (E. coli) expressing both the oxc and frc genes of O. formigenenes. MATERIALS AND METHODS: After the extraction of total RNA from O. formigenes, a reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out using primers synthesized according to the oxc and frc genes reported in GenBank. The cloned cDNA encoding oxalyl-CoA decarboxylase and formyl-CoA transferase was introduced into the pET-22b (+) plasmid vector. The constructs were verified by restriction analysis and DNA sequencing. The plasmid vector containing the cDNA fragment was transformed into competent E. coli BL21 (DE3). The recombinant E. coli was then analyzed using SD-SPAGE for the protein expressions of oxc and frc gene products, and visualized by staining with Coomassie Blue. RESULTS: Restriction enzyme and sequence analyses showed the gene cloned into the pET-22b (+) plasmid vector was identical to the reported oxc and frc genes. After the transformation into the competent E. coli, the SDS-PAGE analysis showed the recombinant E. coli expressed the proteins migrating at 66 and 50KD, which was identical to the reported weight of oxalyl-CoA decarboxylase and formyl-CoA transferase. CONCLISIONS A recombinant E. coli, expressing oxc and frc genes, was successfully produced. Further studies may be necessary to investigate their enzymatic activities on the degradation of oxalate in the development of a new therapeutic strategy for the prevention of stone formation.