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 β-View the MathML source-thiogalactoside-inducible promotor of pSE380 expression vector. The recombinant protein was purified to ∼90% homogeneity and with a yield of ∼9000 units of activity/L of culture, using an efficient one-column procedure. A continuous spectrophotometric assay coupling Pi release to the phosphorolysis of the nucleoside analogue 7-methylinosine (m7Ino) was recently described. Here, we report the steady-state kinetic parameters of the recombinant E. coli PNPase catalyzed reaction with m7Ino and Pi 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 ∼55°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 m7Ino from inosine is described which is safe and cost effective, making the use of this methylated nucleoside in PNPase-coupled Pi assays more attractive