Expression, Purification, and Characterization of Recombinant Purine Nucleoside Phosphorylase from <i>Escherichia coli</i>

Abstract

Recombinant purine nucleoside phosphorylase (PNPase) from <i>Escherichia coli</i> was prepared in high yield in order to facilitate its use in coupled assays to measure the kinetics of phosphate−liberating enzymes. The <i>E. coli</i> enzyme was overexpressed in <i>E. coli</i> by inserting the genomic fragment containing the <i>deoD</i> gene downstream of the isopropyl β−D−thiogalactoside−inducible promotor of pSE380 expression vector. The recombinant protein was purified to about 90% homogeneity and with a yield of about 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 <i>E. coli</i> 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 <i>E. coli</i> protein is active at higher pH values and is stable up to a temperature of about 55&deg;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 attractiv