Production, purification, and luminometric analysis of recombinant Saccharomyces cerevisiae MET3 adenosine triphosphate sulfurylase expressed in Escherichia coli

ATP sulfurylase cDNA from MET3 on chromosome X of Saccharomyces cerevisiae was amplified and cloned, and recombinant ATP sulfurylase was expressed in Escherichia coli. The synthesis of ATP sulfurylase was directed by an expre ssion system that employs the regulatory genes of the luminous bacterium Vi brio fischeri. A soluble, biologically active form was purified to electrop horetic homogeneity from lysates of recombinant E. coli by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The specif ic activity of the purified enzyme was estimated to 140 U/mg. The apparent molecular mass of the recombinant enzyme was determined by gel filtration t o be 470 kDa, which indicates that the active enzyme is an octamer of ident ical subunits (the molecular mass of a single subunit is 59.3 kDa). The ATP sulfurylase activity was monitored in real time by a very sensitive biolum inometric method. (C) 1999 Academic Press.