title: Efficient assembly of recombinant major histocompatibility complex class I molecules with preformed disulfide bonds.
abstract: The expression of major histocompatibility class I (MHC-I) crucially depends upon the binding of appropriate peptides. MHC-I from natural sources are therefore always preoccupied with peptides complicating their purification and analysis. Here, we present an efficient solution to this problem. Recombinant MHC-I heavy chains were produced in Escherichia coli and subsequently purified under denaturing conditions. In contrast to common practice, the molecules were not reduced during the purification. The oxidized MHC-I heavy chain isoforms were highly active with respect to peptide binding. This suggests that de novo folding of denatured MHC-I molecules proceed efficiently if directed by preformed disulfide bond(s). Importantly, these molecules express serological epitopes and stain specific T cells; and they bind peptides specifically. Several denatured MHC-I heavy chains were analyzed and shown to be of a quality, which allowed quantitative analysis of peptide binding. The analysis of the specificity of the several hundred human MHC haplotypes, should benefit considerably from the availability of pre-oxidized recombinant MHC-I.
authors.affiliation: Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: L
authors.initials: L
authors.lastname: Ostergaard Pedersen
authors.forename: M H
authors.initials: MH
authors.lastname: Nissen
authors.forename: N J
authors.initials: NJ
authors.lastname: Hansen
authors.forename: L L
authors.initials: LL
authors.lastname: Nielsen
authors.forename: S L
authors.initials: SL
authors.lastname: Lauenmøller
authors.forename: T
authors.initials: T
authors.lastname: Blicher
authors.forename: A
authors.initials: A
authors.lastname: Nansen
authors.forename: C
authors.initials: C
authors.lastname: Sylvester-Hvid
authors.forename: A R
authors.initials: AR
authors.lastname: Thromsen
authors.forename: S
authors.initials: S
authors.lastname: Buus
mesh_terms: D000818:Animals; D004220:Disulfides; D004926:Escherichia coli / Q000235:genetics; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D010455:Peptides / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology; D001613:beta 2-Microglobulin / Q000378:metabolism
chemical_list: D004220:Disulfides; D015395:Histocompatibility Antigens Class I; D010455:Peptides; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin