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 expression system that employs the regulatory genes of the luminous bacterium Vibrio fischeri. A soluble, biologically active form was purified to electrophoretic homogeneity from lysates of recombinant E. coli by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The specific activity of the purified enzyme was estimated to 140 U/mg. The apparent molecular mass of the recombinant enzyme was determined by gel filtration to be 470 kDa, which indicates that the active enzyme is an octamer of identical 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 bioluminometric method.
Karamohamed
S
S
Department of Biotechnology, The Royal Institute of Technology, Stockholm, SE-100 44, Sweden.
Nilsson
J
J
Nourizad
K
K
Ronaghi
M
M
Pettersson
B
B
Nyrén
P
P
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000254:growth & development; D007700:Kinetics; D008163:Luminescent Measurements; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013430:Sulfate Adenylyltransferase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D013696:Temperature
D018076:DNA, Complementary; D011994:Recombinant Proteins; D013430:Sulfate Adenylyltransferase