Molecular cloning, characterization and purification of ornithine carbamoyltransferase from Mycobacterium bovis BCG. |
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Authors: | Juliano Timm Ingrid Van Rompaey Catherine Tricot Marc Massaer Françoise Haeseleer Alan Fauconnier Victor Stalon Alex Bollen and Paul Jacobs |
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Institution: | (1) Service de Génétique Appliquée, Université Libre de Bruxelles, rue de l'Industrie 24, B-1400 Nivelles, Belgium;(2) Université Libre de Bruxelles et Institut de recherches du Centre d'Enseignement et de Recherches des Industries Alimentaires et Chimiques, Av. E. Gryzon 1, B-1070 Brussels, Belgium;(3) Present address: Unité de Génie Microbiologique, Institut Pasteur, 28 rue du Docteur Roux, F-7015 Paris, France |
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Abstract: | Summary A genomic library of Mycobacterium bovis BCG has been constructed by cloning DNA partially digested with Sau3A into the Escherichia coli expression vector pAS1. The gene coding for ornithine carbamoyltransferase (EC.2.1.3.3 ; OTCase), hereafter referred to as argF, was isolated from the library by complementation of a double argF-argI mutant of E. coli and its sequence was determined. The translation initiation codon used, GTG, was identified by comparing the amino acid sequence deduced from the gene with the N-terminal sequence of the corresponding purified protein. On this basis, the M. bovis BCG OTCase monomer consists of 307 amino acid residues and displays about 44% identity with other OTCases, the most closely related homologue being the anabolic enzyme of Pseudomonas aeruginosa. The native enzyme has an estimated molecular mass of 110 kDa, suggesting a trimeric structure as is the case for most of the anabolic OTCases known from various organisms. |
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Keywords: | Mycobacteria Bacillus Calmette-Guerin (BCG) Ornithine carbamoyltransferase |
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