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Extracellular proteins of Vibrio cholerae: molecular cloning, nucleotide sequence and characterization of the deoxyribonuclease (DNase) together with its periplasmic localization in Escherichia coli K-12
Authors:T Focareta  P A Manning
Affiliation:1. Department of Material and Environmental Chemistry, Graduate School of Engineering, Utsunomiya University, 7-1-2 Yoto, Utsunomiya, Tochigi 321-8585, Japan;2. Kurita Global Technology Center, Kurita Water Industries Ltd., 1-1 Kawada, Nogi-machi, Shimotsuga-gun, Tochigi 329-0105, Japan;1. Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, Leuven, Belgium;2. Department of Gastroenterology, Hepatology and Endocrinology, Center of Internal Medicine, Hannover Medical School, Hannover, Germany;3. MVZ für Lebertransplantation, University Hospital Hamburg Eppendorf, Hamburg, Germany;1. College of Information and Management Science, Henan Agricultural University, 450002 Zhengzhou, China;2. Department of Electrical Engineering and Computer Science, University of Wisconsin–Milwaukee, Milwaukee, WI 53211, USA;1. Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, BP42617, 31326 Castanet Tolosan, France;2. PAPPSO, GQE - Le Moulon, INRA, Univ. Paris-Sud, CNRS, AgroParisTech, Université Paris-Saclay, 91190 Gif-sur-Yvette, France
Abstract:The gene encoding the extracellular DNase of Vibrio cholerae was cloned into Escherichia coli K-12. A maximal coding region of 1.2 kb and a minimal region of 0.6 kb were determined by transposon mutagenesis and deletion analysis. The nucleotide sequence of this region contained a single open reading frame of 690 bp corresponding to a protein of Mr 26,389 with a typical N-terminal signal sequence of 18 aa which, when removed, would give a mature protein of Mr 24,163. This is in good agreement with the size of 24 kDa, calculated directly by Coomassie blue staining following sodium dodecyl sulphate-polyacrylamide gel electrophoresis and indirectly via a DNA-hydrolysis assay. The protein is located in the periplasmic space of E. coli K-12 unlike in V. cholerae where it is excreted into the extracellular medium. The introduction of the DNase gene into a periplasmic (tolA) leaky mutant of E. coli K-12 facilitates the release of the protein, further confirming the periplasmic location.
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