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 similar to 90% homogeneity and with a yield of similar to 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 similar to 55 degreesC 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.