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 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. (C) 2001 Academic Press.