TY - JOUR

T1 - A single-chain fusion molecule consisting of peptide, major histocompatibility gene complex class I heavy chain and beta2-microglobulin can fold partially correctly, but binds peptide inefficiently

AU - Sylvester-Hvid, C

AU - Buus, S

N1 - Keywords: Antigen Presentation; Hemagglutinin Glycoproteins, Influenza Virus; Histocompatibility Antigens Class I; Humans; Models, Immunological; Nickel; Nitrilotriacetic Acid; Peptide Fragments; Protein Binding; Protein Engineering; Protein Folding; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; Sepharose; beta 2-Microglobulin

PY - 1999

Y1 - 1999

N2 - 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 beta2-microglobulin (hbeta2m). 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.

AB - 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 beta2-microglobulin (hbeta2m). 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.

M3 - Journal article

C2 - 10520174

VL - 50

SP - 355

EP - 362

JO - Scandinavian Journal of Immunology, Supplement

JF - Scandinavian Journal of Immunology, Supplement

SN - 0301-6323

IS - 4

ER -