Analysis of the interaction between human interleukin-5 and the soluble domain of its receptor using a surface plasmon resonance biosensor.
A surface plasmon resonance (SPR) biosensor was used to study the interaction of human interleukin-5 (hIL5) with its receptor. IL5 is a major growth factor in the production and activation of eosinophils. The receptor for IL5 is composed of two subunits, alpha and beta. The alpha subunit provides the specificity for IL5 and consists of an extracellular soluble domain, a single transmembrane region and a cytoplasmic tail. We expressed the soluble domain of the human IL5 receptor alpha subunit (shIL5R alpha) and human IL5 (hIL5) in Drosophila. Both hIL5 and shIL5R alpha were immobilized separately through amine groups onto the carboxylated dextran layer of sensor chips of the BIAcore (Pharmacia) SPR biosensor after N-hydroxysuccinimide/carbodiimide activation of the chip surface. Interactions were measured for the complementary macromolecule, either shIL5R alpha or hIL5, in solution. Kinetics of binding of soluble analyte to immobilized ligand were measured and from this the association rate constant, dissociation rate constant and equilibrium dissociation constant (Kd) were derived. With immobilized shIL5R alpha and soluble hIL5, the measured Kd was 2 nM. A similar value was obtained by titration calorimetry. The Kd for Drosophila expressed receptor and IL5 is higher than the values reported for proteins expressed in different systems, likely due to differences in the methods of interaction analysis used or differences in protein glycosylation. Receptor-IL5 binding was relatively pH independent between pH 6.5 and 9.5. Outside this range, the dissociation rate increased with comparatively little increase in association rate.(ABSTRACT TRUNCATED AT 250 WORDS)
Morton
T A
TA
Department of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
Bennett
D B
DB
Appelbaum
E R
ER
Cusimano
D M
DM
Johanson
K O
KO
Matico
R E
RE
Young
P R
PR
Doyle
M
M
Chaiken
I M
IM
D000818:Animals; D015374:Biosensing Techniques; D002151:Calorimetry; D004330:Drosophila; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015848:Interleukin-5 / Q000737:chemistry / Q000378:metabolism*; D008832:Microchemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018123:Receptors, Interleukin / Q000737:chemistry / Q000378:metabolism*; D053649:Receptors, Interleukin-5; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D015848:Interleukin-5; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011993:Recombinant Fusion Proteins