Expression and ligand binding characterization of the beta-subunit (p75) ectodomain of the interleukin-2 receptor.
The baculovirus-mediated eukaryotic insect cell expression system was used to prepare large quantities of the beta-subunit ectodomain of the high-affinity interleukin-2 receptor (IL-2R beta x). We describe the expression, purification, and biophysical characterization of this ligand binding domain. The human cDNA encoding IL-2R beta x was inserted into baculovirus transfer vectors. High titer recombinant baculovirus was produced in Spodoptera frugiperda (Sf9) insect cells, and the viral supernatants were subsequently used to infect monolayers of Trichoplusia ni (High Five) insect cells in serum-free culture. Maximal expression of the recombinant protein excreted into the cell culture supernatants was determined by SDS/PAGE analysis, where a band migrating with an apparent molecular mass of 31 kDa was identified by immunostaining. One-step purification was achieved by affinity chromatography on either a monoclonal antibody (TIC-1) column or an IL-2 column, with a final yield of approximately 5 mg/L of culture supernatant. Interestingly, partial purification was also demonstrated using metal chelate affinity chromatography. Amino-terminal sequence analysis of the protein matched the published sequence. Both equilibrium sedimentation analysis and gel filtration chromatography indicated that IL-2R beta x remains monomeric. Deconvolution of far-UV circular dichroism (CD) spectra indicated the predominant secondary structural element to be beta-sheet, consistent with structural analysis and predictions for other members of the hematopoietic receptor family. A dissociation constant (Kd) for IL-2R beta x in solution of 5.3 x 10(-7) M was calculated from competitive receptor binding assays.(ABSTRACT TRUNCATED AT 250 WORDS)
Sana
T R
TR
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755.
Wu
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Z
Smith
K A
KA
Ciardelli
T L
TL
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001667:Binding, Competitive; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D007376:Interleukin-2 / Q000378:metabolism; D009036:Moths; D010446:Peptide Fragments / Q000096:biosynthesis / Q000378:metabolism; D015375:Receptors, Interleukin-2 / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
D007376:Interleukin-2; D010446:Peptide Fragments; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins