Purification and ligand binding of a soluble class I major histocompatibility complex molecule consisting of the first three domains of H-2K^d fused to β₂-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-2K^d are fused to β₂-microglobulin (β₂-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2K^d (SC-K^d) 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-2K^d monoclonal antibody SF1.1.1.1. We tested the ability of SC-K^d to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2K^d-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-2K^d heavy chain in infected cells in the absence of co-expressed β₂-microglobulin. SC-K^d expressed in insect cells did not require additional mouse β₂-m to bind the photoprobe, indicating that the covalently attached β₂-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-K^d molecule was unaffected by the addition of exogenous β₂-m. This is in contrast to H-2K^dQ₁₀, a soluble H-2K^d molecule in which β₂-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 β₂-m. The binding of the probe to SC-K^d was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2K^d molecule.