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 histocompatibili ty complex molecules HLA-DRB50101 and DRB1*0101 were expressed separa tely in Escherichia coli. The cytoplasmic and membrane-spanning domain s of both chains were replaced by oligohistidine tags to allow purific ation by metal chelate chromatography. The recombinant proteins were r efolded to peptide-free, water-soluble heterodimers by removal of majo r amounts of detergents and concomitant reoxidiation of disulfide bond s. Correct conformation was documented by three criteria: (a) affinity binding experiments using the antibody L243, which is known to recogn ize a conformational epitope formed only by correctly associated heter odimers; (b) specific binding of peptides to the refolded molecules; ( c) recognition of peptides bound to refolded HLA-DR molecules by T-cel ls as reflected by Ca2+ influx into T cells and production of interfer on-gamma. The refolding reaction did not absolutely depend on the pres ence of peptides. The yield of peptide-free heterodimers was 3.0%. How ever, the yield of refolded heterodimer was increased to 10% if refold ing was performed in the presence of antigenic peptides.