Role of chain pairing for the production of functional soluble IA major histocompatibility complex class II molecules.
Structural studies of cellular receptor molecules involved in immune recognition require the production of large quantities of the extracellular domains of these glycoproteins. The murine major histocompatibility complex (MHC) class II-restricted response has been extensively studied by functional means, but the engineering and purification of the native, empty form of the most-studied murine MHC class II molecule, IA, has been difficult to achieve. IA molecules, which are the murine equivalent of human histocompatibility leukocyte antigen-DQ molecules, have a low efficiency of chain pairing, which results in poor transport to the cell surface and in the appearance of mixed isotype pairs. We have engineered soluble IA molecules whose pairing has been forced by the addition of leucine zipper peptide dimers at their COOH-terminus. The molecules are secreted "empty" into the extracellular medium and can be loaded with single peptide after purification. These IA molecules have been expressed in milligram quantity for crystallization as well as for activation of T cells and measurement of MHC class II-T cell receptor interactions.
Scott
C A
CA
Scripps Research Institute, La Jolla, California 92037, USA.
Garcia
K C
KC
Carbone
F R
FR
Wilson
I A
IA
Teyton
L
L
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D017931:DNA Primers; D018076:DNA, Complementary; D004331:Drosophila melanogaster; D006683:HLA-DQ Antigens / Q000276:immunology; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D016350:Leucine Zippers; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D013601:T-Lymphocytes / Q000276:immunology*; D013917:Thrombin; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D006683:HLA-DQ Antigens; D000949:Histocompatibility Antigens Class II; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; D013917:Thrombin