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A Single‐Chain Fusion Molecule Consisting of Peptide, Major Histocompatibility Gene Complex Class I Heavy Chain and β 2 ‐Microglobulin Can Fold Partially Correctly, but Binds Peptide Inefficiently
Author(s) -
Christina Sylvester-Hvid,
Line Nielsen,
Nils Jakob Vest Hansen,
Lars Østergaard Pedersen,
Søren Buus
Publication year - 1999
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1046/j.1365-3083.1999.00601.x
Subject(s) - major histocompatibility complex , beta 2 microglobulin , ctl* , mhc class i , recombinant dna , mhc restriction , epitope , peptide , microbiology and biotechnology , biology , cd8 , chemistry , biochemistry , antigen , cytotoxic t cell , in vitro , gene , genetics , immunology
The function of major histocompatibility complex class I (MHC‐I) molecules is to sample peptides from the intracellular environment and present these peptides to CD8 + cytotoxic T lymphocytes (CTL). We have attempted to develop a general approach to produce large amounts of pure and active recombinant MHC‐I molecules. A convenient source of MHC‐I molecules would be a valuable tool in structural and biochemical analysis of MHC‐I, and in experiments using MHC‐I molecules to enable specific manipulations of experimental and physiological CTL responses. Here we describe the generation of a recombinant murine MHC‐I molecule, which could be produced in large amounts in bacteria. The recombinant MHC‐I protein was expressed as a single molecule (PepSc) consisting of the antigenic peptide linked to the MHC‐I heavy chain and further linked to human β 2 ‐microglobulin (hβ 2 m). The PepSc molecule was denatured, extracted, purified and folded using a recently developed in vitro reiterative refolding strategy. This led to the formation of soluble, recombinant MHC‐I molecules, which migrated as monomers of the expected size when submitted to non‐reducing sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Serological analysis revealed the presence of some, but not all, MHC‐I‐specific epitopes. Biochemically, PepSc could bind peptide, however, rather ineffectively. We suggest that a partially correctly refolded MHC‐I has been obtained.