A generic method for the production of cell lines expressing high levels of 7-transmembrane receptors.
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.
Koller
K J
KJ
Affymax Research Institute, Palo Alto, California 94304, USA. Kerry_Koller@qmgates.affymax.com
Whitehorn
E A
EA
Tate
E
E
Ries
T
T
Aguilar
B
B
Chernov-Rogan
T
T
Davis
A M
AM
Dobbs
A
A
Yen
M
M
Barrett
R W
RW
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006388:Hemagglutinins / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D006388:Hemagglutinins; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins