Soluble mouse major histocompatibility complex class II molecules produced in Drosophila cells.
We have exploited Drosophila melanogaster Schneider cells and compatible inducible expression vectors to produce large amounts of secreted major histocompatibility complex (MHC) class II molecules (I-Ed). A simple two-step purification protocol was developed. In the first step, recombinant molecules were enriched using a monoclonal anti-class II 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 alpha beta heterodimers. Further analysis shows that the recombinant MHC class II molecules are devoid of endogenous peptides and, therefore, homogeneous peptide/MHC 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 II molecules, together with various forms of soluble T cell receptor (TcR) proteins, provide valuable tools to analyze the molecular details of TcR/antigen recognition.
Wallny
H J
HJ
Basel Institute for Immunology, Switzerland.
Sollami
G
G
Karjalainen
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004331:Drosophila melanogaster / Q000166:cytology; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006825:Hybridomas; D008213:Lymphocyte Activation; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D012995:Solubility; D013601:T-Lymphocytes / Q000276:immunology
D000911:Antibodies, Monoclonal; D000949:Histocompatibility Antigens Class II; C024138:I-E-antigen; D010455:Peptides; D011993:Recombinant Fusion Proteins