TY - JOUR

T1 - Efficient assembly of recombinant major histocompatibility complex class I molecules with preformed disulfide bonds.

AU - Ostergaard Pedersen, L

AU - Nissen, Mogens Holst

AU - Hansen, N J

AU - Nielsen, L L

AU - Lauenmøller, S L

AU - Blicher, T

AU - Nansen, A

AU - Sylvester-Hvid, C

AU - Thomsen, Allan Randrup

AU - Buus, S

N1 - Keywords: Animals; Disulfides; Escherichia coli; Histocompatibility Antigens Class I; Humans; Hydrogen-Ion Concentration; Mice; Peptides; Protein Folding; Recombinant Proteins; T-Lymphocytes; beta 2-Microglobulin

PY - 2001

Y1 - 2001

N2 - 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.

AB - 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.

M3 - Journal article

C2 - 11592075

VL - 31

SP - 2986

EP - 2996

JO - European Journal of Immunology

JF - European Journal of Immunology

SN - 0014-2980

IS - 10

ER -