Expression and ligand binding assays of soluble cytokine receptor-immunoglobulin fusion proteins.
We have developed a cloning vector for the expression of type I cytokine receptor, NO, extracellular domain (ECD)-mouse IgG1 Fc fusion proteins. The vector has a versatile polylinker that allows in-frame cloning of the receptor ECD with the mouse IgG1 sequence to encode a receptor ECD-IgG1 fusion construct. The receptor-IgG1 fusion proteins are transiently expressed in useful amounts following transfection of the expression vector into COS7 cells and G418 selection. The mouse IgG1 portion of the fusion protein provides a universal handle for purification on an affinity matrix and detection by anti-mouse IgG antibodies in ELISA or Western blot formats. The expressed receptor ECD-IgG1 fusion proteins bind their cognate ligands. In order to demonstrate that the fusion proteins have similar ligand binding affinities as the native receptors, the affinity constants (Kd) for EPOR, TNFR, IL-4R, and IL-6R-IgG1 fusion proteins were measured by surface plasmon resonance and shown to be in good agreement with published values. The TNFR-IgG1 fusion protein was employed in a demonstration of a novel ELISA format for detecting cytokine receptor binding to cytokine.
Brown
S J
SJ
Chugai Biopharmaceuticals, Inc., 6275 Nancy Ridge Drive, San Diego, California, 92121, USA. sbrown@chugaibio.com
Becherer
K A
KA
Blumeyer
K
K
Kautzer
C
C
Axelrod
F
F
Le
H
H
McConnell
S J
SJ
Whalley
A
A
Spinella
D G
DG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015374:Biosensing Techniques; D019556:COS Cells; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D018121:Receptors, Cytokine / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D008024:Ligands; D018121:Receptors, Cytokine; D011993:Recombinant Fusion Proteins