Refolding of human class II major histocompatibility complex molecules isolated from Escherichia coli. Assembly of peptide-free heterodimers and increased refolding-yield in the presence of antigenic peptide.
The alpha- and beta-chains of the heterodimeric major histocompatibility complex molecules HLA-DRB5*0101 and DRB1*0101 were expressed separately in Escherichia coli. The cytoplasmic and membrane-spanning domains of both chains were replaced by oligohistidine tags to allow purification by metal chelate chromatography. The recombinant proteins were refolded to peptide-free, water-soluble heterodimers by removal of major amounts of detergents and concomitant reoxidiation of disulfide bonds. Correct conformation was documented by three criteria: (a) affinity binding experiments using the antibody L243, which is known to recognize a conformational epitope formed only by correctly associated heterodimers; (b) specific binding of peptides to the refolded molecules; (c) recognition of peptides bound to refolded HLA-DR molecules by T-cells as reflected by Ca2+ influx into T-cells and production of interferon-gamma. The refolding reaction did not absolutely depend on the presence of peptides. The yield of peptide-free heterodimers was 3.0%. However, the yield of refolded heterodimer was increased to 10% if refolding was performed in the presence of antigenic peptides.
Max-Planck-Institut für Psychiatrie, Abteilung Neuroimmunologie, Martinsried, Germany.
J
J
Stöckel
E
E
Meinl
C
C
Hahnel
J
J
Malotka
R
R
Seitz
K
K
Drexler
H
H
Wekerle
K
K
Dornmair
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006684:HLA-DR Antigens / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology
D006684:HLA-DR Antigens; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011994:Recombinant Proteins