Two-step purification of tag-free norovirus-like particles from silkworm larvae (Bombyx mori).
Protein Expr Purif
; 190: 106010, 2022 02.
Article
em En
| MEDLINE
| ID: mdl-34737040
ABSTRACT
Recombinantly expressed VP1 of norovirus self-assembled and formed norovirus-like particles (NoV-LPs). This native VP1 was expressed using the Bombyx mori nucleopolyhedrovirus (BmNPV) expression system in silkworm larva. NoV-LPs were collected from silkworm fat body lysate by density gradient centrifugation. To improve the purity of the NoV-LP, the proteins were further purified using immobilized metal affinity chromatography based on the surface exposed side chain of histidine residues. The additional purification led to a highly purified virus-like particle (VLP). The morphology and size of the purified VLPs were examined using a transmission electron microscope, and dynamic light scattering revealed a monodispersed spherical morphology with a diameter of 34 nm. The purified product had a purity of >90% with a recovery yield of 48.7% (equivalent to 930 µg) from crude lysate, obtained from seven silkworm larvae. In addition, the purified VLP could be recognized by antibodies against GII norovirus in sandwich enzyme-linked immunosorbent assay, which indicated that the silkworm-derived VLP is biologically functional as a NoV-LP in its native state, is structurally correct, and exerts its biological function. Our results suggest that the silkworm-derived NoV-LP may be useful for subsequent applications, such as in a vaccine platform. Moreover, the silkworm-based expression system is known for its robustness, facile up-scalability, and relatively low expense compared to insect cell systems.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Bombyx
/
Proteínas Virais de Fusão
/
Norovirus
/
Partículas Artificiais Semelhantes a Vírus
Limite:
Animals
Idioma:
En
Revista:
Protein Expr Purif
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Japão