A single-chain fusion molecule consisting of peptide, major histocompatibility gene complex class I heavy chain and beta(2)-microglobulin can fold partially correctly, but binds peptide inefficiently

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 deve lop a general approach to produce large amounts of pure and active recombin ant MHC-I molecules. A convenient source of MHC-I molecules would be a valu able tool in structural and biochemical analysis of MHC-I, and in experimen ts using MHC-I molecules to enable specific manipulations of experimental a nd physiological CTL responses. Here we describe the generation of a recomb inant murine MHC-I molecule, which could be produced in large amounts in ba cteria. The recombinant MHC-I protein was expressed as a single molecule (P epSc) consisting of the antigenic peptide linked to the MHC-I heavy chain a nd further linked to human beta(2)-microglobulin (h beta(2)m). The PepSc mo lecule was denatured, extracted, purified and folded using a recently devel oped in vitro reiterative refolding strategy. This led to the formation of soluble, recombinant MHC-I molecules, which migrated as monomers of the exp ected size when submitted to non-reducing sodium dodecyl sulphate-polyacryl amide gel electrophoresis (SDS-PAGE). Serological analysis revealed the pre sence of some, but not all, MHC-I-specific epitopes. Biochemically, PepSc c ould bind peptide, however, rather ineffectively. We suggest that a partial ly correctly refolded MHC-I has been obtained.