Article: article from journal or magazin.
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.
The Journal of biological chemistry
Publication types: Journal Article Publication Status: ppublish
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 SF126.96.36.199. 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.
Adenovirus E1A Proteins/genetics, Adenovirus E1A Proteins/metabolism, Adenoviruses, Human/genetics, Adenoviruses, Human/metabolism, Amino Acid Sequence, Animals, Antigens, Protozoan/metabolism, Baculoviridae/genetics, Base Sequence, Binding Sites, Binding, Competitive, Cell Line, Chromatography, Affinity, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Genes, MHC Class I, Genes, Viral, H-2 Antigens/genetics, H-2 Antigens/isolation & purification, Insects, Kinetics, Macromolecular Substances, Mice, Models, Structural, Molecular Sequence Data, Molecular Weight, Oligodeoxyribonucleotides, Oligopeptides/pharmacology, Plasmodium berghei/metabolism, Protein Binding, Protein Sorting Signals/genetics, Protein Sorting Signals/metabolism, Protozoan Proteins, Recombinant Fusion Proteins/isolation & purification, Recombinant Fusion Proteins/metabolism, Transfection, beta 2-Microglobulin/genetics, beta 2-Microglobulin/isolation & purification
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