High level expression and purification of the Epstein-Barr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin.
Authors
Salek-Ardakani, ShahramStuart, 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.orgIssue Date
2002-01-07
Metadata
Show full item recordAbstract
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 CytokineJournal
CytokineDOI
10.1006/cyto.2001.0990PubMed ID
11886166Type
ArticleLanguage
enISSN
1043-4666ae974a485f413a2113503eed53cd6c53
10.1006/cyto.2001.0990
Scopus Count
Collections
Related articles
- Purification and characterization of decorin core protein expressed in Escherichia coli as a maltose-binding protein fusion.
- Authors: Hering TM, Kollar J, Huynh TD, Varelas JB
- Issue date: 1996 Aug 15
- Overexpression of bacterio-opsin in Escherichia coli as a water-soluble fusion to maltose binding protein: efficient regeneration of the fusion protein and selective cleavage with trypsin.
- Authors: Chen GQ, Gouaux JE
- Issue date: 1996 Mar
- Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme.
- Authors: Martinez A, Knappskog PM, Olafsdottir S, Døskeland AP, Eiken HG, Svebak RM, Bozzini M, Apold J, Flatmark T
- Issue date: 1995 Mar 1
- Characterization of purified recombinant Bet v 1 with authentic N-terminus, cloned in fusion with maltose-binding protein.
- Authors: Spangfort MD, Ipsen H, Sparholt SH, Aasmul-Olsen S, Larsen MR, Mørtz E, Roepstorff P, Larsen JN
- Issue date: 1996 Nov
- Expression of pig heart mitochondrial NADP-dependent isocitrate dehydrogenase in Escherichia coli.
- Authors: Soundar S, Jennings GT, McAlister-Henn L, Colman RF
- Issue date: 1996 Nov