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

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

Author Pereira, NAG Google Scholar
Juliano, M. A. Google Scholar
Carmona, A. K. Google Scholar
Sturrock, E. D. Google Scholar
Kotwal, G. J. Google Scholar
Lahiri, D. K. Google Scholar
Institution Univ Cape Town
Universidade Federal de São Paulo (UNIFESP)
Abstract Kaposi'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.
Keywords cloning
Kaposi sarcoma herpesvirus (KSHV)
open reading frame (ORF) 4
yeast
Pichia pastoris
vaccinia virus complement control protein (VCP)
Kaposi's sarcoma complement control protein (KCP)
Language English
Date 2005-01-01
Published in Natural Products and Molecular Therapy. New York: New York Acad Sciences, v. 1056, p. 388-404, 2005.
ISSN 0077-8923 (Sherpa/Romeo, impact factor)
Publisher New York Acad Sciences
Extent 388-404
Origin http://dx.doi.org/10.1196/annals.1352.019
Access rights Closed access
Type Article
Web of Science ID WOS:000235433100033
URI http://repositorio.unifesp.br/handle/11600/28109

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