Reconstitution of class I MHC molecules expressed in E. coli and complexed with single antigenic peptides.
The HLA-Cw3 heavy chain has been expressed at high level as insoluble protein aggregates in E. coli. The protein aggregates dissolved in strong denaturant solution were efficiently reconstituted by removal of denaturant in the presence of an HLA-Cw3 binding peptide (FAM) and beta 2m. The reconstituted HLA-Cw3/FAM protein binds specifically to a p58 natural killer cell inhibitory receptor, a natural ligand. The HLA-A2 molecule has also been reconstituted in complex with either of a peptide from myelin associated glycoprotein (MAG) or a peptide from the GAG protein of human immunodeficiency virus. The HLA-A2/MAG protein crystallized under the identical conditions as HLA-A2 purified from human lymphoblastoid cells. The reconstitution method has yielded an abundant supply of HLA molecules complexed with single antigenic peptides, and may be of general utility in reconstituting any class I MHC molecules. However, the HLA molecules could not be reconstituted either without a peptide or with an irrelevant peptide. Using this property, the reconstitution method could be used to determine whether a peptide is restricted/bound to certain class I MHC molecule.
Kim
J
J
Department of Microbiology, Yonsei University College of Medicine, Seoul, Korea.
Kim
M Y
MY
Chwae
Y J
YJ
Park
J H
JH
D003460:Crystallization; D004926:Escherichia coli; D015789:HLA-A2 Antigen / Q000302:isolation & purification; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D010455:Peptides / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D015789:HLA-A2 Antigen; D015395:Histocompatibility Antigens Class I; D010455:Peptides; D011994:Recombinant Proteins