title: Expression of the bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli.
abstract: The fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (EC 2.7.105/EC 3.1.3.46) was expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase. The protein was efficiently expressed (i) as a fusion protein that starts at the T7 major capsid protein initiation site in a pET expression vector and (ii) as a protein that starts within the bisphosphatase sequence by translation reinitiation. Both proteins have similar properties. The protein was purified to homogeneity by anion-exchange chromatography and gel filtration. The purified fructose-2,6-bisphosphatase domain was active and no 6-phosphofructo-2-kinase activity was found associated with it. In contrast to the dimeric bifunctional enzyme, the fructose-2,6-bisphosphatase domain behaved as a monomer of 30 kDa. The turnover number and kinetic properties of the separate bisphosphatase domain were similar to those of the bisphosphatase of the bifunctional enzyme, including the ability to form a phosphoenzyme intermediate. These results support the hypothesis that the rat liver enzyme consists of two independent domains and is a member of a class of enzymes formed by gene fusion.
authors.affiliation: Department of Physiology and Biophysics, State University of New York, Stony Brook 11794.
authors.forename: A
authors.initials: A
authors.lastname: Tauler
authors.forename: A H
authors.initials: AH
authors.lastname: Rosenberg
authors.forename: A
authors.initials: A
authors.lastname: Colosia
authors.forename: F W
authors.initials: FW
authors.lastname: Studier
authors.forename: S J
authors.initials: SJ
authors.lastname: Pilkis
mesh_terms: D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis*; D010770:Phosphotransferases / Q000096:biosynthesis*; D051381:Rats
chemical_list: D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases