PMID:11437593
| Citation |
Lee, J, Filosa, S, Bonvin, J, Guyon, S, Aponte, RA and Turnbull, JL (2001) Expression, purification, and characterization of recombinant purine nucleoside phosphorylase from Escherichia coli. Protein Expr. Purif. 22:180-8 |
|---|---|
| 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 beta-d-thiogalactoside-inducible promotor of pSE380 expression vector. The recombinant protein was purified to approximately 90% homogeneity and with a yield of approximately 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 approximately 55 degrees 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 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. |
| Links |
PubMed Online version:10.1006/prep.2001.1437 |
| Keywords |
Actinomycetales/enzymology; Circular Dichroism; Cloning, Molecular; Enzyme Stability; Escherichia coli/enzymology; Escherichia coli/genetics; Genetic Vectors/chemical synthesis; Hydrogen-Ion Concentration; Inosine/analogs & derivatives; Inosine/chemical synthesis; Inosine/metabolism; Kinetics; Molecular Weight; Osmolar Concentration; Phosphates/metabolism; Protein Structure, Secondary; Purine-Nucleoside Phosphorylase/biosynthesis; Purine-Nucleoside Phosphorylase/chemistry; Purine-Nucleoside Phosphorylase/genetics; Purine-Nucleoside Phosphorylase/isolation & purification; Recombinant Proteins/biosynthesis; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Substrate Specificity |
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