title: Expression, purification, and characterization of recombinant purine nucleoside phosphorylase from Escherichia coli.
abstract: Recombinant purine nucleoside phosphorylase (PNPase) from Escherichia coli was prepared in high yield in order to facilitate its use in coupled assays to measure the kinetics of phosphate-liberating enzymes. The E. coli enzyme was overexpressed in E. coli by inserting the genomic fragment containing the deoD gene downstream of the isopropyl beta-d-thiogalactoside-inducible promotor of pSE380 expression vector. The recombinant protein was purified to approximately 90% homogeneity and with a yield of approximately 9000 units of activity/L of culture, using an efficient one-column procedure. A continuous spectrophotometric assay coupling P(i) release to the phosphorolysis of the nucleoside analogue 7-methylinosine (m(7)Ino) was recently described. Here, we report the steady-state kinetic parameters of the recombinant E. coli PNPase catalyzed reaction with m(7)Ino and P(i) as substrates and compare these parameters with those of a bacterial PNPase commercially available for use in coupled assays. Under the assay conditions described, the recombinant E. coli protein is active at higher pH values and is stable up to a temperature of approximately 55 degrees C and following multiple freeze-thaw cycles. It is activated by high ionic strength but loses some activity following dialysis or concentration under pressure. Finally, a new procedure for the synthesis of m(7)Ino from inosine is described which is safe and cost effective, making the use of this methylated nucleoside in PNPase-coupled P(i) assays more attractive.
authors: Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, H3G-1M8, Canada.
authors: J
authors: J
authors: Lee
authors: S
authors: S
authors: Filosa
authors: J
authors: J
authors: Bonvin
authors: S
authors: S
authors: Guyon
authors: R A
authors: RA
authors: Aponte
authors: J L
authors: JL
authors: Turnbull
mesh_terms: D000192:Actinomycetales / Q000201:enzymology; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005822:Genetic Vectors / Q000138:chemical synthesis; D006863:Hydrogen-Ion Concentration; D007288:Inosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009994:Osmolar Concentration; D010710:Phosphates / Q000378:metabolism; D017433:Protein Structure, Secondary; D011683:Purine-Nucleoside Phosphorylase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D013379:Substrate Specificity
chemical_list: D010710:Phosphates; D011994:Recombinant Proteins; C034907:7-methylinosine; D007288:Inosine; D011683:Purine-Nucleoside Phosphorylase