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

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    Authors
    Salek-Ardakani, Shahram
    Stuart, Amanda D
    Arrand, Jane E
    Lyons, Steve
    Arrand, John R
    Mackett, Mike
    Affiliation
    Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Withington, Manchester, M20 4BX, UK. ssalek@liai.org
    Issue Date
    2002-01-07
    
    Metadata
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    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.
    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
    URI
    http://hdl.handle.net/10541/84337
    DOI
    10.1006/cyto.2001.0990
    PubMed ID
    11886166
    Type
    Article
    Language
    en
    ISSN
    1043-4666
    ae974a485f413a2113503eed53cd6c53
    10.1006/cyto.2001.0990
    Scopus Count
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    All Paterson Institute for Cancer Research

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