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          Institute: MPI für medizinische Forschung     Collection: Abteilung Biomolekulare Mechanismen     Display Documents



ID: 567805.0, MPI für medizinische Forschung / Abteilung Biomolekulare Mechanismen
Expression, Purification, and Characterization of Recombinant Purine Nucleoside Phosphorylase from <i>Escherichia coli</i>
Translation of Title:Expression, Purification, and Characterization of Recombinant Purine Nucleoside Phosphorylase from <i>Escherichia coli</i>
Authors:Lee, John; Filosa, Serena; Bonvin, Julie; Guyon, Sebastien; Aponte, Raphael A.; Turnbull, Joanne L.
Language:English
Date of Publication (YYYY-MM-DD):2001-07-02
Title of Journal:Protein Expression and Purification
Journal Abbrev.:Protein Expression Purif.
Volume:22
Issue / Number:2
Start Page:180
End Page:188
Review Status:Peer-review
Audience:Experts Only
Intended Educational Use:No
Abstract / Description:Recombinant purine nucleoside phosphorylase (PNPase) from <i>Escherichia coli</i> was prepared in high yield in order to facilitate its use in coupled assays to measure the kinetics of phosphate−liberating enzymes. The <i>E. coli</i> enzyme was overexpressed in <i>E. coli</i> by inserting the genomic fragment containing the <i>deoD</i> gene downstream of the isopropyl β−D−thiogalactoside−inducible promotor of pSE380 expression vector. The recombinant protein was purified to about 90% homogeneity and with a yield of about 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 <i>E. coli</i> 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 <i>E. coli</i> protein is active at higher pH values and is stable up to a temperature of about 55&deg;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
Last Change of the Resource (YYYY-MM-DD):--
External Publication Status:published
Document Type:Article
Communicated by:Wulf Kaiser
Affiliations:MPI für medizinische Forschung/Abteilung Biomolekulare Mechanismen
MPI für medizinische Forschung/Abteilung Biomolekulare Mechanismen/Molecular chaperones
Identifiers:LOCALID:7097
URI:http%3A%2F%2Fwww.sciencedirect.com%2Fscience%3F_ob...
URI:http%3A%2F%2Fdx.doi.org%2F10.1006%2Fprep.2001.1437
URI:http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F11437...
DOI:10.1006%2Fprep.2001.1437
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