Immunoaffinity Purification of the Glycosylated Extracellular Fragment of Mouse Plexin A2 Produced in a Mammalian Expression System

Methods Mol Biol. 2017:1493:57-72. doi: 10.1007/978-1-4939-6448-2_4.

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.

Keywords: Crystallographic analysis; Glycosylated protein; High-density cell culture; Immunoaffinity purification; Mammalian expression system; Stable expression.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatography, Affinity / methods*
  • Crystallography, X-Ray
  • Electrophoresis, Polyacrylamide Gel
  • Glycosylation
  • HEK293 Cells
  • Humans
  • Mice
  • Mutation
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / isolation & purification*
  • Receptors, Cell Surface / chemistry
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / isolation & purification*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification

Substances

  • Nerve Tissue Proteins
  • Plxna2 protein, mouse
  • Receptors, Cell Surface
  • Recombinant Proteins