In vitro assay for the Rap GTPase-activating protein activity of the purified cytoplasmic domain of plexin

Heath G. Pascoe, Yuxiao Wang, Xuewu Zhang

Research output: Contribution to journalArticle

Abstract

Plexins are cell surface receptors that bind semaphorins and regulate essential processes such as axon guidance and angiogenesis. The cytoplasmic regions of plexins contain a functionally essential GTPase-activating protein (GAP) domain, which initiates downstream signaling by specifically inactivating the Rap GTPase. Here we describe the methods for expression and purification of the plexin cytoplasmic region in E. coli, and characterization of its GAP activity using a photometric assay. We also provide a protocol for measuring GAP activity of single-chain constructs with Rap covalently linked to the plexin cytoplasmic region.

Original languageEnglish (US)
Pages (from-to)107-118
Number of pages12
JournalMethods in Molecular Biology
Volume1493
DOIs
StatePublished - 2017

Keywords

  • GAP
  • GAP activity assay
  • Plexin
  • Rap
  • Semaphorin

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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