Cloning and expression of a functionally active truncated N-glycosylated KSHVORF4/KCP/Kaposica in the methylotrophic yeast Pichia pastoris

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dc.contributor.authorPereira, NAG
dc.contributor.authorJuliano, M. A.
dc.contributor.authorCarmona, A. K.
dc.contributor.authorSturrock, E. D.
dc.contributor.authorKotwal, G. J.
dc.contributor.authorLahiri, D. K.
dc.contributor.institutionUniv Cape Town
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.date.accessioned2016-01-24T12:37:36Z
dc.date.available2016-01-24T12:37:36Z
dc.date.issued2005-01-01
dc.description.abstractKaposi's sarcoma herpesvirus (KSHV) is a typical DNA virus that is associated with a number of proliferative diseases including Kaposi's sarcoma. the KSHV open reading frame (ORF) 4 encodes a complement regulatory protein (Kaposi complement control protein, KCP) that binds complement components and inhibits the complement-mediated lysis of cells infected by the virus, thus providing a strategy for evasion of the host complement system. Based on primary sequence analysis and comparison with other functionally and structurally similar proteins, oligonucleotide primers were designed to amplify by polymerase chain reaction (PCR) three regions of the predicted ORF 4 from human herpes virus-8 (HHV-8) DNA isolated from a primary effusion lymphoma cell line. the PCR products were inserted by ligation into the expression vector pPIC9 to generate three recombinant plasmids for heterologous expression in the yeast, Pichia pastoris, to produce separately the four N-terminal sushi domains (KCP-S, small), KCP protein lacking the putative transmembrane-binding domain (KCP-M, medium), and the full-length protein (KCP-F, full). Expression of the viral proteins was confirmed by SDSPAGE, immunologic detection, and Western blot analyses using a rabbit polyclonal antibody directed against a selected peptide region that is common to all three recombinant KCPs. KCP-S directly from expression media could inhibit complement-mediated lysis of sensitized sheep erythrocytes by approximately 60% in a hemolysis assay. This result confirms previous reports that recombinant KCP is twice as efficient in inhibiting the classic pathway-mediated lysis of erythrocytes than is the vaccinia virus complement control protein, which also contains four sushi domains.en
dc.description.affiliationUniv Cape Town, Fac Hlth Sci, Div Med Virol, Inst Infect Dis & Mol Med, ZA-7925 Cape Town, South Africa
dc.description.affiliationUniversidade Federal de São Paulo, Escola Paulista Med, Div Nephrol, Dept Biophys, BR-04044020 São Paulo, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Escola Paulista Med, Div Nephrol, Dept Biophys, BR-04044020 São Paulo, Brazil
dc.description.sourceWeb of Science
dc.format.extent388-404
dc.identifierhttp://dx.doi.org/10.1196/annals.1352.019
dc.identifier.citationNatural Products and Molecular Therapy. New York: New York Acad Sciences, v. 1056, p. 388-404, 2005.
dc.identifier.doi10.1196/annals.1352.019
dc.identifier.issn0077-8923
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/28109
dc.identifier.wosWOS:000235433100033
dc.language.isoeng
dc.publisherNew York Acad Sciences
dc.relation.ispartofNatural Products and Molecular Therapy
dc.rightsinfo:eu-repo/semantics/restrictedAccess
dc.subjectcloningen
dc.subjectKaposi sarcoma herpesvirus (KSHV)en
dc.subjectopen reading frame (ORF) 4en
dc.subjectyeasten
dc.subjectPichia pastorisen
dc.subjectvaccinia virus complement control protein (VCP)en
dc.subjectKaposi's sarcoma complement control protein (KCP)en
dc.titleCloning and expression of a functionally active truncated N-glycosylated KSHVORF4/KCP/Kaposica in the methylotrophic yeast Pichia pastorisen
dc.typeinfo:eu-repo/semantics/article
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