Korean J Urol.  2007 Feb;48(2):206-211. 10.4111/kju.2007.48.2.206.

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

• Park YH
• Jeong BC
• Kwak C
• Oh JE
• Kim BS
• Kim EC
• Kim HH

• KMID: 2139762
• DOI: http://doi.org/10.4111/kju.2007.48.2.206

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.

Keyword

Oxalobacter formigenes; Escherichia coli; oxc and frc

MeSH Terms

Clone Cells
Databases, Nucleic Acid
DNA, Complementary
Electrophoresis, Polyacrylamide Gel
Escherichia coli*
Escherichia*
Oxalates
Oxalobacter formigenes*
Plasmids
RNA
Sequence Analysis
Sequence Analysis, DNA
Transferases
DNA, Complementary
Oxalates
RNA
Transferases

Figure

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  Fig. 1 The coding sequence of the oxc gene.

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  Fig. 2 The coding sequence of the frc gene.

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  Fig. 3 The structure of pET-22b (+) vector, including the T7 promotor (A) and the structure of the constructed oxc/frc expression vector [named pET-22b(+) - oxc/frc] (B).

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  Fig. 4 SDS-PAGE analysis of the induced oxc gene recombinant E. coli using Coomassie blue staining. The new band located at 66KD was shown after the induction of recombinant E. coli compared with the control (non-recombinant E. coli). Standard MW: standard molecular weight.

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  Fig. 5 SDS-PAGE analysis of the induced frc gene recombinant E. coli using Coomassie blue staining. The new band located at 50KD was shown after the induction of recombinant E. coli compared with the control (non-recombinant E. coli). Standard MW: standard molecular weight.

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