Oxalobacter formigenes의 oxc와 frc 유전자를 발현하는 재조합 대장균의 개발
Development of Recombinant Escherichia coli expressing oxc and frc Gene of Oxalobacter formigenes
박용현, 정병창, 곽철, 오지은, 김봉섭, 김현회, 김의종,
소속 상세정보
박용현 ( Park Yong-Hyun )
서울대학교 의과대학 비뇨기과학교실
정병창 ( Jeong Byong-Chang )
서울대학교 의과대학 비뇨기과학교실
곽철 ( Kwak Cheol )
서울대학교 의과대학 비뇨기과학교실
오지은 ( Oh Ji-Eun )
서울대학교 의과대학 비뇨기과학교실
김봉섭 ( Kim Bong-Sub )
서울대학교 의과대학 비뇨기과학교실
김현회 ( Kim Hyeon-Hoe )
서울대학교 의과대학 비뇨기과학교실
김의종 ( Kim Eui-Chong )
서울대학교 의과대학 검사의학교실
KMID : 0358320070480020206
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 & 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.
Conclusion: 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. (Korean J Urol 2007;48:206-211)
키워드
Oxalobacter formigenes;Escherichia coli; oxc and frc
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