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- Title
A Single-Chain Fusion Molecule Consisting of Peptide, Major Histocompatibility Gene Complex Class I Heavy Chain and β[sub 2]-Microglobulin Can Fold Partially Correctly, but Binds Peptide Inefficiently.
- Authors
Sylvester-Hvid; Nielsen; Hansen; Pedersen; Buus
- Abstract
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[sup +] 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[sub 2]-microglobulin (hβ[sub 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.
- Subjects
HISTOCOMPATIBILITY antigens; PEPTIDES
- Publication
Scandinavian Journal of Immunology, 1999, Vol 50, Issue 4
- ISSN
0300-9475
- Publication type
Article
- DOI
10.1046/j.1365-3083.1999.00601.x