CLONING AND EXPRESSION OF E.COLI ORNITHINE TRANSCARBAMYLASE GENE, argl = 대장균 오니틴 트랜스 카바밀라제 유전자 클로닝 및 발현

다국어 초록 (Multilingual Abstract)

E. coli ornithine transcarbamylase is the enzyme which catalyzes the citrullin biosynthesis from ornithine and carbamyl phosphate. To facilitate the purification of enzyme which will be used for many purposes, cloning and expression of E. coli argI gene, ornithine transcarbamylase gene, have been conducted. argI has been amplified from genomic DNA of E. coli DH5a strain by PCR method, and cloned in prokaryotic expression vector, pKK223-3. The enzyme expressed has been purified by salt fractionation, heat denaturation and affinity chromatography. The result of SDS PAGE shows a single band of the purified enzyme. Kinetic data for expressed enzyme give almost same results as those of the wild type enzyme. Vmax of the enzyme 1.0x105 min-¹, and Kms of ornithine and carbamyl phosphate are 0.35 mM and 0.06 mM, respectively. On the other hand, TB2 cell has been obtained by plasmid curing. This strain does not have argI gene and resulting ornithine transcarbamylase activity. The strain has been prepared for site-directed mutagenesis work.