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KMID : 0903519950380020118
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Journal of the Korean Society of Agricultural Chemistry and Biotechnology
1995 Volume.38 No. 2 p.118 ~ p.122
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Cloning and Expression of Escherichia coli Ornithine Transcarbamylase Gene , argⅠ
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Abstract
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Escherichia Coli ornithine transcarbamylase is the enzyme which catalyzes the L-citrulline biosynthesis from L-ornithine and carbamyl phosphate. To facilitate the purification of enzyme which will be used for many biochemical studies such as structure and function relationships and catalytic mechanisms, the cloning and expression of E. coli argI gene for ornithine transcarbamylase was conducted. argI was amplified from genomic DNA of E. coli strain of DH5α, by polymerization chain reaction (PCR) method. The amplified argI gene was ligated to the prokaryotic expression vector pKK223-3 and used for transformation of E. coli TB2 which was deficient of ornithine transcarbamylase. The over-produced enzyme by the tnansformant was purified by ammonium sulfate fractionation, heat denaturation and affinity chromatography. The result of SDS denaturation gel electrophoresis for the purified enzyme showed a single band of about 38 kDa of ornithine transcarbamylase. Kinetic data for the expressed enzyme gave almost the scone values as those of the wild type enzyme. The k_(cat), of the enzyme was 1.0×10^5 min^(-1) and K_ms for ornithine and carbamyl phosphate were 0.35 mM and 0.06 mM, respectively.
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