Soluble mouse major histocompatibility complex class I1 molecules produced in Drosophila cells
Basel lnstitut for Immunology
We have exploited Drosophilu melunoguster Schneider cells and compatible inducible expression vectors to produce large amounts of secreted major histocompatibility complex (MHC) class I1 molecules (I-Ed). A simple two-step purification protocol was developed. In the first step, recombinant molecules were enriched using a monoclonal anti-class I1 antibody column followed by a nickel chelate column which further purified and concentrated the recombinant protein to several mg/ml. Characterization of the purified material indicates that the molecules are correctly assembled into ct(3 heterodimers. Further analysis shows that the recombinant MHC class II molecules are devoid of endogenous peptides and, therefore, homogenous peptideMHC complexes could be prepared by adding exogenous I-Ed-specific peptides at slightly acidic pH. Upon peptide addition, molecules underwent a conformational change into a more compact form revealed by gel filtration analysis. In addition, the peptide/MHC complexes were biologically active. As little as 10 ng of these complexes coated on plastic from a 100 ng/ml solution were sufficient to trigger antigen-specific T cell hybridomas. These MHC class 11 molecules, together with various forms of soluble T cell receptor (TcR) proteins, provide valuable tools to analyze the molecular details of TcR/antigen recognition.