A generic method for the production of cell lines expressing high levels of 7-transmembrane receptors

Anal Biochem. 1997 Jul 15;250(1):51-60. doi: 10.1006/abio.1997.2190.

Abstract

G-protein-coupled or 7-transmembrane receptors (7TMRs) are often studied after heterologous expression in mammalian cells such as COS-7, CHO-K1, or HEK-293s. In this paper, we describe the development of a rapid and generic method for producing stable Chinese hamster ovary cell lines expressing high levels of recombinant 7TMRs by N-terminal tagging these proteins with the hemagglutinin (HA) sequence. To illustrate the broad applicability of this technique, we have presented data from cell lines expressing a glycoprotein hormone receptor for follicle-stimulating hormone (FSHR), CXC- (CXCR-2), and CC-chemokine (CCR-1) receptors and peptide receptors from the somatostatin (SSTR1, 2, 5) and neuropeptide Y (NPY-Y2, -Y4 Rs) families. Typically, cell lines with a receptor density of 1 to 15 pmol/mg protein are produced with this method. The presence of the HA tag does not adversely affect the binding or functional activity of the receptors.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal
  • CHO Cells
  • Cricetinae
  • GTP-Binding Proteins / biosynthesis*
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / metabolism
  • Gene Expression*
  • Genetic Vectors / genetics
  • Hemagglutinins / genetics
  • Molecular Sequence Data
  • Plasmids
  • Protein Sorting Signals
  • Receptors, Cell Surface / biosynthesis*
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Transfection

Substances

  • Antibodies, Monoclonal
  • Hemagglutinins
  • Protein Sorting Signals
  • Receptors, Cell Surface
  • Recombinant Proteins
  • GTP-Binding Proteins