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.
J Biol Chem
; 269(47): 29571-8, 1994 Nov 25.
Article
em En
| MEDLINE
| ID: mdl-7961943
ABSTRACT
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.
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Coleções:
01-internacional
Contexto em Saúde:
3_ND
Base de dados:
MEDLINE
Assunto principal:
Antígenos HLA-DR
/
Dobramento de Proteína
/
Escherichia coli
Limite:
Humans
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
1994
Tipo de documento:
Article