A simple method to purify recombinant HCV core protein expressed in Pichia pastoris for obtaining virus-like particles and producing monoclonal antibodies.
Protein Expr Purif
; 183: 105864, 2021 07.
Article
em En
| MEDLINE
| ID: mdl-33677084
ABSTRACT
In this study, we describe an optimized method of obtaining virus-like particles (VLPs) of the recombinant hepatitis C virus (HCV) core protein (HCcAg) expressed in yeast cells (Pichia pastoris), which can be used for the construction of diagnostic test systems and vaccine engineering. The described simplified procedure was developed to enable in vitro self-assembly of HCcAg molecules into VLPs during protein purification. In brief, the HCcAg protein was precipitated from yeast cell lysates with ammonium sulfate and renatured by gel filtration on Sephadex G-25 under reducing conditions. VLPs were self-assembled after the removal of the reducing agent by gel filtration on Sephadex G-25. Protein purity and specificity were evaluated by SDS-PAGE and immunoblotting analysis. The molecular mass of VLPs and their relative quantity were measured by HPLC, followed by confirmation of VLPs production and estimation of their shape and size by transmission electron microscopy. As a result, we obtained recombinant HCcAg preparation (with ~90% purity) in the form of VLPs and monomers, which has been used to produce hybridomas secreting monoclonal antibodies (mAbs) against HCcAg.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Vacinas contra Hepatite Viral
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Proteínas do Core Viral
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Hepacivirus
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Anticorpos Anti-Hepatite C
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Saccharomycetales
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Anticorpos Monoclonais Murinos
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Vacinas de Partículas Semelhantes a Vírus
Limite:
Animals
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article