Expression, purification, and characterization of recombinant purine nucleoside phosphorylase from Escherichia coli.
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.
Lee
J
J
Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, H3G-1M8, Canada.
Filosa
S
S
Bonvin
J
J
Guyon
S
S
Aponte
R A
RA
Turnbull
J L
JL
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
D010710:Phosphates; D011994:Recombinant Proteins; C034907:7-methylinosine; D007288:Inosine; D011683:Purine-Nucleoside Phosphorylase