Expression of recombinant HLA-DR2 molecules. Replacement of the hydrophobic transmembrane region by a leucine zipper dimerization motif allows the assembly and secretion of soluble DR alpha beta heterodimers.
Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers that present peptides on the surface of antigen presenting cells to T cells. Soluble HLA-DR2 molecules were expressed for structural and functional characterization of the MHC/peptide/T cell receptor recognition unit. The alpha and beta chains of DR2 (encoded by the DRA, DRB1*1501 genes) did not assemble in mammalian or insect cell lines when the transmembrane regions of one or both chains were truncated. The hydrophobic transmembrane regions of DRalpha and DRbeta facilitate assembly of the heterodimer and were therefore replaced by the leucine zipper dimerization motifs from the transcription factors Fos and Jun, which assemble as a soluble, tightly packed coiled coil structure. The DRalpha-Fos and DRbeta-Jun constructs were expressed in a methyltrophic yeast, Pichia pastoris, using the alpha-mating factor secretion signal to direct expression to the secretory pathway. DR alphabeta heterodimers were purified from supernatants using an antibody specific for the DR alphabeta heterodimer. Kinetic and quantitative peptide binding experiments demonstrated that recombinant DR2 molecules were efficiently loaded with an antigenic peptide. Soluble DR2 molecules can be used to define structural aspects of the MHC/peptide/T cell receptor interaction and to study the signals induced by T cell receptor recognition of soluble DR2.peptide complexes.
Kalandadze
A
A
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Galleno
M
M
Foncerrada
L
L
Strominger
J L
JL
Wucherpfennig
K W
KW
D000595:Amino Acid Sequence; D001665:Binding Sites; D015870:Gene Expression; D015802:HLA-DR2 Antigen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016350:Leucine Zippers; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins; D012995:Solubility
D015802:HLA-DR2 Antigen; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins