Immunoaffinity purification of the glycosylated extracellular fragment of mouse Plexin A2 produced in a mammalian expression system

Terukazu Nogi, Emiko Mihara, Norihisa Yasui, Junichi Takagi

Research output: Contribution to journalArticle

Abstract

Plexins are type I membrane proteins that function as receptors for semaphorins. All of the known plexins contain a large globular domain, termed the sema domain, in the N-terminal extracellular region, which interacts with semaphorins during signal transduction. Here, we describe procedures for protein production and purification that we utilized in the crystallographic study of the mouse Plexin A2 (mPlxnA2) extracellular fragment, including the sema domain. A mutant mammalian cell line, HEK293S GnTI−, was used as an expression host for the production of a crystallizable-quality mPlxnA2 fragment, which contains several N-glycosylation sites and disulfide bonds.

Original languageEnglish
Pages (from-to)57-72
Number of pages16
JournalMethods in Molecular Biology
Volume1493
DOIs
Publication statusPublished - 2017

Fingerprint

Semaphorins
varespladib methyl
Glycosylation
Disulfides
Signal Transduction
Membrane Proteins
Cell Line
Proteins
plexin

Keywords

  • Crystallographic analysis
  • Glycosylated protein
  • High-density cell culture
  • Immunoaffinity purification
  • Mammalian expression system
  • Stable expression

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Immunoaffinity purification of the glycosylated extracellular fragment of mouse Plexin A2 produced in a mammalian expression system. / Nogi, Terukazu; Mihara, Emiko; Yasui, Norihisa; Takagi, Junichi.

In: Methods in Molecular Biology, Vol. 1493, 2017, p. 57-72.

Research output: Contribution to journalArticle

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