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Expression, purification, and characterization of recombinant purine nucleoside phosphorylase from Escherichia coli

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Aponte,  Raphael A.
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;

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Expression, purification, and characterization of recombinant purine nucleoside phosphorylase from Escherichia coli.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0019-8E42-A
Abstract
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.