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 lig and 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 vira l supernatants were subsequently used to infect monolayers of Trichopl usia ni (High Five) insect cells in serum-free culture. Maximal expres sion of the recombinant protein excreted into the cell culture superna tants was determined by SDS/ PAGE analysis, where a band migrating wit h an apparent molecular mass of 31 kDa was identified by immunostainin g. One-step purification was achieved by affinity chromatography on ei ther a monoclonal antibody (TIC-1) column or an IL-2 column, with a fi nal yield of approximately 5 mg/L of culture supernatant. Interestingl y, partial purification was also demonstrated using metal chelate affi nity chromatography. Amino-terminal sequence analysis of the protein m atched 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 in dicated 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 (K-d) f or IL-2R beta x in solution of 5.3 X 10(-7) M was calculated from comp etitive receptor binding assays. These results indicate that the IL-2 receptor beta-subunit lacking both the transmembrane and cytoplasmic d omains can bind IL-2 in solution with 1:1 stoichiometry.