First archaeal inorganic polyphosphate/ATP-dependent NAD kinase, from hyperthermophilic archaeon Pyrococcus horikoshii: cloning, expression, and characterization.

ABSTRACT: The gene (PH1074) encoding the NAD kinase of the hyperthermophilic archaeon Pyrococcus horikoshii was identified in the genome database, cloned, and functionally expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment at 90 degrees C for 20 min and one successive HiTrap affinity chromatography step. The purified enzyme was easily precipitated by dialysis against phosphate buffer without NaCl and imidazole and was usually stored in buffer containing 0.5 M NaCl and 0.5 M imidazole to avoid precipitation. The molecular mass of the active enzyme was determined to be 145 kDa by a gel filtration method, and the enzyme was composed of a tetramer of 37-kDa subunits. The archaeal enzyme utilized several nucleoside triphosphates, such as GTP, CTP, UTP, and

SUBMITTER: Sakuraba H 

PROVIDER: S-EPMC1183369 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature