Expression and Purification of Class 7 Semaphorin and Its PlexinC1 Receptor Using Baculovirus-Mediated Mammalian Cell Gene Transduction.
Methods Mol Biol
; 1493: 41-56, 2017.
Article
em En
| MEDLINE
| ID: mdl-27787841
ABSTRACT
Semaphorins and their receptor plexins are large glycoproteins that are difficult to express using regular recombinant methods, and the widely used E. coli and baculovirus-insect cell systems have been inadequate for semaphorins and plexins which contain a large number of domains and are heavily modified by glycosylation. Here, we describe the expression of class 7 semaphorin (Sema7A) and the extracellular domain of its receptors PlexinC1, using the baculovirus-mediated mammalian cell gene transduction (BacMam) method. A robust mammalian cell expression gene cassette, including a highly efficient secretion signal peptide, is introduced into the baculovirus which subsequently enters mammalian cells for efficient expression in suspension cell culture. Large amount of high-infectivity BacMam viruses are needed for infecting suspended mammalian cells in large scale, to generate semaphorin and plexin proteins at an amount sufficient for binding experiments and crystallographic studies. The inclusion of serum in expression ensures the robustness of cell culture, but introduces substantial amount of contaminant proteins interfering with immobilized metal ion affinity purification, which can be overcome with a two-step purification scheme.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Receptores Virais
/
Transdução Genética
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Baculoviridae
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Receptores de Superfície Celular
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Semaforinas
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Estados Unidos