title: Cloning, expression, and efficient purification in Escherichia coli of a halophilic nucleoside diphosphate kinase from the moderate halophile Halomonas sp. #593.
abstract: Most typical halophilic enzymes from extremely halophilic archaea require high concentrations of salt for their activity and stability. These enzymes are inactive in Escherichia coli unless refolded in the presence of salts in vitro. In this report, we describe cloning of the ndk gene of nucleoside diphosphate kinase from a moderately halophilic eubacterium and overexpression of the protein in E. coli as an N-terminal hexa-His fusion to facilitate its purification on Ni-NTA affinity resin. We demonstrate evidence that the protein is properly folded and exhibits the same specific activity and stability as the native protein from Halomonas cells.
authors.affiliation: Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Yonezawa
authors.forename: Hiroko
authors.initials: H
authors.lastname: Tokunaga
authors.forename: Matsujiro
authors.initials: M
authors.lastname: Ishibashi
authors.forename: Satoru
authors.initials: S
authors.lastname: Taura
authors.forename: Masao
authors.initials: M
authors.lastname: Tokunaga
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics; D020632:Halomonas / Q000201:enzymology* / Q000235:genetics; D006639:Histidine; D008969:Molecular Sequence Data; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012492:Salts / Q000494:pharmacology
chemical_list: D011993:Recombinant Fusion Proteins; D012492:Salts; D006639:Histidine; D009701:Nucleoside-Diphosphate Kinase