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
en 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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Vacunas contra Hepatitis Viral
/
Proteínas del Núcleo Viral
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Hepacivirus
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Anticuerpos contra la Hepatitis C
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Saccharomycetales
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Anticuerpos Monoclonales de Origen Murino
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Vacunas de Partículas Similares a Virus
Límite:
Animals
Idioma:
En
Revista:
Protein Expr Purif
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2021
Tipo del documento:
Article