Expression of Plant Receptor Kinases in Tobacco BY-2 Cells

Methods Mol Biol. 2017:1621:29-35. doi: 10.1007/978-1-4939-7063-6_3.

Abstract

Although more than 600 single-transmembrane receptor kinase genes have been found in the Arabidopsis genome, only a few of them have known physiological functions, and even fewer plant receptor kinases have known specific ligands. Ligand-binding analysis must be operated using the functionally expressed receptor form. However, the relative abundance of native receptor kinase molecules in the plasma membrane is often quite low. Here, we present a method for stable and functional expression of plant receptor kinases in tobacco BY-2 cells that allows preparation of microsomal fractions containing the receptor. This procedure provides a sufficient amount of receptor proteins while maintaining its ligand-binding activities.

Keywords: HaloTag; Ligand binding; Peptide ligand; Receptor kinase.

MeSH terms

  • Agrobacterium / genetics
  • Agrobacterium / metabolism
  • Arabidopsis / enzymology
  • Arabidopsis / genetics*
  • Arabidopsis Proteins / biosynthesis
  • Arabidopsis Proteins / genetics*
  • Azides / chemistry
  • Cell Communication
  • Cell Membrane / chemistry
  • Cell Membrane / enzymology
  • Electrophoresis, Polyacrylamide Gel / methods
  • Fluorescent Dyes / chemistry
  • Gene Expression*
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Heterocyclic Compounds, 3-Ring / chemistry
  • Ligands
  • Microsomes / chemistry
  • Microsomes / enzymology
  • Nicotiana / cytology
  • Nicotiana / enzymology
  • Nicotiana / genetics*
  • Plant Cells / enzymology*
  • Protein Binding
  • Protein Serine-Threonine Kinases / biosynthesis
  • Protein Serine-Threonine Kinases / genetics*
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Rhodamines
  • Salicylates / chemistry
  • Signal Transduction
  • Staining and Labeling / methods
  • Transformation, Genetic

Substances

  • Arabidopsis Proteins
  • Azides
  • Fluorescent Dyes
  • Heterocyclic Compounds, 3-Ring
  • Ligands
  • Recombinant Fusion Proteins
  • Rhodamines
  • Salicylates
  • tetramethylrosamine
  • 4-azidosalicylic acid
  • BAM1 protein, Arabidopsis
  • Protein Serine-Threonine Kinases