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

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

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ProteinExpressionPurification_22_2001_180.pdf (Any fulltext), 137KB
 
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ProteinExpressionPurification_22_2001_180.pdf
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 Creators:
Lee, John, Author
Filosa, Serena, Author
Bonvin, Julie, Author
Guyon, Sebastien, Author
Aponte, Raphael A.1, Author              
Turnbull, Joanne L., Author
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1Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society, ou_1497700              

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 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.

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Language(s): eng - English
 Dates: 2001-07-02
 Publication Status: Published in print
 Pages: -
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 Rev. Type: Peer
 Identifiers: DOI: 10.1006/prep.2001.1437
Other: 7097
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