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.
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.
Sylvester-Hvid
C
C
Institute of Medical Microbiology and Immunology, University of Copenhagen, Denmark.
Nielsen
L L
LL
Hansen
N J
NJ
Pedersen
L O
LO
Buus
S
S
D017951:Antigen Presentation; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018448:Models, Immunological; D009532:Nickel; D009571:Nitrilotriacetic Acid; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering; D017510:Protein Folding; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012685:Sepharose; D001613:beta 2-Microglobulin / Q000235:genetics / Q000378:metabolism*
C122115:HA (255-262); D019267:Hemagglutinin Glycoproteins, Influenza Virus; D015395:Histocompatibility Antigens Class I; D010446:Peptide Fragments; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin; D009532:Nickel; D012685:Sepharose; D009571:Nitrilotriacetic Acid