Expression and purification of class 7 semaphorin and its Plexinc1 receptor using baculovirus-mediated mammalian cell gene transduction

Xiaoyan Chen, Po Han Chen, Xiaolin He*

*Corresponding author for this work

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)41-56
Number of pages16
JournalMethods in Molecular Biology
Volume1493
DOIs
StatePublished - Jan 1 2017

Fingerprint

Semaphorins
Baculoviridae
Genes
Cell Culture Techniques
Protein Sorting Signals
Glycosylation
Insects
Suspensions
Glycoproteins
Proteins
Metals
Ions
Escherichia coli
Viruses
Gene Expression
Serum

Keywords

  • BacMam
  • Baculovirus
  • Cell-surface receptor
  • Glycoprotein
  • Mammalian cell expression
  • Plexin
  • Semaphorin
  • Suspension mammalian cell culture

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

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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.",
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Expression and purification of class 7 semaphorin and its Plexinc1 receptor using baculovirus-mediated mammalian cell gene transduction. / Chen, Xiaoyan; Chen, Po Han; He, Xiaolin.

In: Methods in Molecular Biology, Vol. 1493, 01.01.2017, p. 41-56.

Research output: Contribution to journalArticle

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