High level expression and purification of the Epstein-Barr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin.

2.50
Hdl Handle:
http://hdl.handle.net/10541/84337
Title:
High level expression and purification of the Epstein-Barr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin.
Authors:
Salek-Ardakani, Shahram; Stuart, Amanda D; Arrand, Jane E; Lyons, Steve; Arrand, John R; Mackett, Mike
Abstract:
To characterize the structural and functional properties of viral interleukin 10 (vIL-10), its cDNA was cloned into the bacterial expression vector pMAL-c2, which directs the synthesis of the inserted gene as a fusion protein with maltose binding protein (MBP). The MBP-vIL-10 fusion protein was expressed in Escherichia coli and purified from cell lysates using amylose resin chromatography. Viral interleukin 10 (IL-10) was released from the fusion protein by cleavage with the proteolytic enzyme factor Xa. We show that vIL-10 will bind to heparin and use this property to purify vIL-10 from factor Xa cleaved products and trace contaminants using heparin agarose chromatography. A simple one-step procedure is described for the removal of endotoxins from heavily contaminated vIL-10 preparations. The protocol exploits the high binding affinity of MBP for amylose resin or vIL-10 for heparin and the ability of Triton-X114 to dissociate endotoxins from proteins. The biological activity of purified vIL-10 was demonstrated through its ability to inhibit interferon gamma (IFN-gamma) production by mitogen activated peripheral blood mononuclear cells and to down-regulate HLA-class II expression on activated monocytes/macrophages. The availability of an efficient expression and purification strategy for vIL-10 together with appropriate assays will contribute to a greater understanding of how vIL-10 has evolved to retain and modify those activities of cellular IL-10 best suited for Epstein-Barr virus (EBV)'s specialized niche within the host.
Affiliation:
Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Withington, Manchester, M20 4BX, UK. ssalek@liai.org
Citation:
High level expression and purification of the Epstein-Barr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin. 2002, 17 (1):1-13 Cytokine
Journal:
Cytokine
Issue Date:
7-Jan-2002
URI:
http://hdl.handle.net/10541/84337
DOI:
10.1006/cyto.2001.0990
PubMed ID:
11886166
Type:
Article
Language:
en
ISSN:
1043-4666
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorSalek-Ardakani, Shahramen
dc.contributor.authorStuart, Amanda Den
dc.contributor.authorArrand, Jane Een
dc.contributor.authorLyons, Steveen
dc.contributor.authorArrand, John Ren
dc.contributor.authorMackett, Mikeen
dc.date.accessioned2009-10-16T11:11:31Z-
dc.date.available2009-10-16T11:11:31Z-
dc.date.issued2002-01-07-
dc.identifier.citationHigh level expression and purification of the Epstein-Barr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin. 2002, 17 (1):1-13 Cytokineen
dc.identifier.issn1043-4666-
dc.identifier.pmid11886166-
dc.identifier.doi10.1006/cyto.2001.0990-
dc.identifier.urihttp://hdl.handle.net/10541/84337-
dc.description.abstractTo characterize the structural and functional properties of viral interleukin 10 (vIL-10), its cDNA was cloned into the bacterial expression vector pMAL-c2, which directs the synthesis of the inserted gene as a fusion protein with maltose binding protein (MBP). The MBP-vIL-10 fusion protein was expressed in Escherichia coli and purified from cell lysates using amylose resin chromatography. Viral interleukin 10 (IL-10) was released from the fusion protein by cleavage with the proteolytic enzyme factor Xa. We show that vIL-10 will bind to heparin and use this property to purify vIL-10 from factor Xa cleaved products and trace contaminants using heparin agarose chromatography. A simple one-step procedure is described for the removal of endotoxins from heavily contaminated vIL-10 preparations. The protocol exploits the high binding affinity of MBP for amylose resin or vIL-10 for heparin and the ability of Triton-X114 to dissociate endotoxins from proteins. The biological activity of purified vIL-10 was demonstrated through its ability to inhibit interferon gamma (IFN-gamma) production by mitogen activated peripheral blood mononuclear cells and to down-regulate HLA-class II expression on activated monocytes/macrophages. The availability of an efficient expression and purification strategy for vIL-10 together with appropriate assays will contribute to a greater understanding of how vIL-10 has evolved to retain and modify those activities of cellular IL-10 best suited for Epstein-Barr virus (EBV)'s specialized niche within the host.en
dc.language.isoenen
dc.subject.meshATP-Binding Cassette Transporters-
dc.subject.meshAmylose-
dc.subject.meshAnimals-
dc.subject.meshCarrier Proteins-
dc.subject.meshChromatography-
dc.subject.meshCloning, Molecular-
dc.subject.meshDNA, Complementary-
dc.subject.meshDown-Regulation-
dc.subject.meshElectrophoresis, Polyacrylamide Gel-
dc.subject.meshEndotoxins-
dc.subject.meshEscherichia coli-
dc.subject.meshEscherichia coli Proteins-
dc.subject.meshFactor Xa-
dc.subject.meshHeparin-
dc.subject.meshHerpesvirus 4, Human-
dc.subject.meshHumans-
dc.subject.meshInterferon-gamma-
dc.subject.meshInterleukin-10-
dc.subject.meshLeukocytes, Mononuclear-
dc.subject.meshMonosaccharide Transport Proteins-
dc.subject.meshOpen Reading Frames-
dc.subject.meshPolyethylene Glycols-
dc.subject.meshRats-
dc.subject.meshRecombinant Fusion Proteins-
dc.subject.meshSepharose-
dc.titleHigh level expression and purification of the Epstein-Barr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin.en
dc.typeArticleen
dc.contributor.departmentPaterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Withington, Manchester, M20 4BX, UK. ssalek@liai.orgen
dc.identifier.journalCytokineen
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