High level expression and purification of the Epstein-Barr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin.
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.
Salek-Ardakani
Shahram
S
Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Withington, Manchester, M20 4BX, UK. ssalek@liai.org
Stuart
Amanda D
AD
Arrand
Jane E
JE
Lyons
Steve
S
Arrand
John R
JR
Mackett
Mike
M
D018528:ATP-Binding Cassette Transporters; D000688:Amylose / Q000737:chemistry; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry; D002845:Chromatography; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D016753:Interleukin-10 / Q000096:biosynthesis* / Q000737:chemistry*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D017830:Octoxynol; D016366:Open Reading Frames; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D018076:DNA, Complementary; D004731:Endotoxins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D016753:Interleukin-10; D011092:Polyethylene Glycols; D007371:Interferon-gamma; D017830:Octoxynol; D006493:Heparin; D000688:Amylose; D012685:Sepharose; C010615:Nonidet P-40; D015951:Factor Xa