High level expression and purification of the Epstein-Barr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin.
Salek-Ardakani, Shahram Stuart, Amanda D Arrand, Jane E Lyons, Steve Arrand, John R Mackett, Mike
Salek-Ardakani, Shahram
Stuart, Amanda D
Arrand, Jane E
Lyons, Steve
Arrand, John R
Mackett, Mike
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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.
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Date
2002-01-07
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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