Purification and ligand binding of a soluble class I major histocompatibility complex molecule consisting of the first three domains of H-2Kd fused to beta 2-microglobulin expressed in the baculovirus-insect cell system.
A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2Kd-restricted Plasmodium berghei circumsporozoite protein (P.b. CS) peptide 253-260 (YIPSAEKI), a probe that we had previously shown to be unable to bind to the H-2Kd heavy chain in infected cells in the absence of co-expressed beta 2-microglobulin. SC-Kd expressed in insect cells did not require additional mouse beta 2-m to bind the photoprobe, indicating that the covalently attached beta 2-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-Kd molecule was unaffected by the addition of exogenous beta 2-m. This is in contrast to H-2KdQ10, a soluble H-2Kd molecule in which beta 2-m is noncovalently bound to the soluble heavy chain, whose ability to bind the photoaffinity probe is greatly enhanced in the presence of an excess of exogenous beta 2-m. The binding of the probe to SC-Kd was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2Kd molecule.
Godeau
F
F
Unité de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale U 277, Paris, France.
Luescher
I F
IF
Ojcius
D M
DM
Saucier
C
C
Mottez
E
E
Cabanie
L
L
Kourilsky
P
P
D017500:Adenovirus E1A Proteins / Q000235:genetics / Q000378:metabolism; D000260:Adenoviruses, Human / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005805:Genes, MHC Class I; D005814:Genes, Viral; D006183:H-2 Antigens / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007313:Insecta; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides / Q000494:pharmacology; D010962:Plasmodium berghei / Q000378:metabolism; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D001613:beta 2-Microglobulin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D017500:Adenovirus E1A Proteins; D000953:Antigens, Protozoan; D006183:H-2 Antigens; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin; C041595:circumsporozoite protein, Protozoan