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

Eur J Immunol. 2001 Oct;31(10):2986-96. doi: 10.1002/1521-4141(2001010)31:10<2986::aid-immu2986>3.0.co;2-r.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Disulfides
  • Escherichia coli / genetics
  • Histocompatibility Antigens Class I / biosynthesis*
  • Histocompatibility Antigens Class I / chemistry
  • Histocompatibility Antigens Class I / immunology
  • Humans
  • Hydrogen-Ion Concentration
  • Mice
  • Peptides / metabolism
  • Protein Folding
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / immunology
  • T-Lymphocytes / immunology
  • beta 2-Microglobulin / metabolism


  • Disulfides
  • Histocompatibility Antigens Class I
  • Peptides
  • Recombinant Proteins
  • beta 2-Microglobulin