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

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High level expression and purification of the Epstein-Barr virus-encoded cytokine viral interleukin-10: Efficient removal of endotoxin. / Salek-Ardakani, S; Stuart, AD; Arrand, JE; Lyons, S; Arrand, John; Mackett, M.

In: Cytokine, Vol. 17, No. 1, 01.01.2002, p. 1-13.

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Salek-Ardakani, S ; Stuart, AD ; Arrand, JE ; Lyons, S ; Arrand, John ; Mackett, M. / High level expression and purification of the Epstein-Barr virus-encoded cytokine viral interleukin-10: Efficient removal of endotoxin. In: Cytokine. 2002 ; Vol. 17, No. 1. pp. 1-13.

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@article{c41a136825234a058e629aee1d397063,
title = "High level expression and purification of the Epstein-Barr virus-encoded cytokine viral interleukin-10: Efficient removal of endotoxin",
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.",
author = "S Salek-Ardakani and AD Stuart and JE Arrand and S Lyons and John Arrand and M Mackett",
year = "2002",
month = jan,
day = "1",
doi = "10.1006/cyto.2001.0990",
language = "English",
volume = "17",
pages = "1--13",
journal = "Cytokine",
issn = "1043-4666",
publisher = "Elsevier",
number = "1",

}

RIS

TY - JOUR

T1 - High level expression and purification of the Epstein-Barr virus-encoded cytokine viral interleukin-10: Efficient removal of endotoxin

AU - Salek-Ardakani, S

AU - Stuart, AD

AU - Arrand, JE

AU - Lyons, S

AU - Arrand, John

AU - Mackett, M

PY - 2002/1/1

Y1 - 2002/1/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0036058131&partnerID=8YFLogxK

U2 - 10.1006/cyto.2001.0990

DO - 10.1006/cyto.2001.0990

M3 - Article

C2 - 11886166

VL - 17

SP - 1

EP - 13

JO - Cytokine

JF - Cytokine

SN - 1043-4666

IS - 1

ER -