Development of Yvrk Gene-Recombinant E. coli Degrading Oxalate.
10.4111/kju.2009.50.10.1022
- Author:
Byong Chang JEONG
1
;
Yong Hyun PARK
;
Hyeon Hoe KIM
Author Information
1. Department of Urology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
- Publication Type:Original Article
- Keywords:
Oxalates;
Hyperoxaluria;
Bacillus subtilis
- MeSH:
Ampicillin;
Arabinose;
Bacillus subtilis;
Bacteria;
Base Sequence;
Blotting, Western;
Calcium Oxalate;
Carboxy-Lyases;
Clone Cells;
DNA;
Escherichia coli;
Hyperoxaluria;
Oxalates;
Plasmids;
Polymerase Chain Reaction;
Sequence Analysis, DNA
- From:Korean Journal of Urology
2009;50(10):1022-1026
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
PURPOSE: Recently, the whole DNA sequence of Bacillus subtilis (B. subtilis) was identified, revealing the existence of the YvrK gene encoding a 43 kD oxalate decarboxylase (OXDC), which degrades oxalate by a simple pathway. The objective of this study was to develop recombinant Escherichia coli (E. coli) expressing the Yvrk gene from B. subtilis. MATERIALS AND METHODS: After the extraction of total DNA from B. subtilis, the YvrK gene was cloned by polymerase chain reaction. The cloned DNA encoding OXDC was inserted into the pBAD/gIII-A vector, downstream of the L-arabinose promotor. The plasmid vector was transformed into TOP 10 E. coli, and the transformants were selected with ampicillin. The recombinant E. coli, named pBy, was then analyzed by DNA sequencing and Western blot. To evaluate the oxalate-degrading function of pBy, pBy was cultured in LB broth containing oxalate, and then the amount of oxalate in the medium was assessed. The oxalate-degrading activity of homogenates of pBy was evaluated. RESULTS: DNA sequencing showed the successful transformation of the YvrK gene into TOP 10 E. coli. Western blot analyses showed that pBy expressed OXDC. pBy removed oxalate during the overnight culture in oxalate-containing LB broth, and the homogenate of pBy degraded 90% of oxalate under acidic conditions. CONCLUSIONS: A recombinant E. coli expressing the YvrK gene was successfully produced. The bacteria showed potent oxalate-degrading activity. The results of this study will provide a solution to the treatment of calcium oxalate stones and hyperoxaluria, for which there are few medical treatment modalities.
