A simple method to purify recombinant HCV core protein expressed in Pichia pastoris for obtaining virus-like particles and producing monoclonal antibodies
| Autor: | Segal Ol, Alexey Nagel, Viacheslav F Lavrov, Zhanna I. Andreeva-Kovalevskaya, Yulia Tarakanova, A. A. Pechelyulko, Alexander S. Solonin, D. A. Dmitriev, Yulia S Massino, Olga Sokolova, Alexandre Dmitriev |
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| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
Viral Hepatitis Vaccines medicine.drug_class viruses Hepacivirus Monoclonal antibody complex mixtures 01 natural sciences law.invention Pichia pastoris 03 medical and health sciences Antibodies Monoclonal Murine-Derived Mice Virus-like particle law 010608 biotechnology Protein purification medicine Animals Vaccines Virus-Like Particle 030304 developmental biology 0303 health sciences Mice Inbred BALB C biology Molecular mass Chemistry Viral Core Proteins Hepatitis C Antibodies biology.organism_classification Yeast Biochemistry Sephadex Saccharomycetales Recombinant DNA Female Biotechnology |
| Zdroj: | Protein expression and purification. 183 |
| ISSN: | 1096-0279 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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