Abstract
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.
Highlights
Part of the Biochemistry Commons, Immunity Commons, Immunology of Infectious Disease Commons, and the Medical Immunology Commons
Purification and ligand binding of a soluble class I MHC molecule consisting of the first three domains of H-2Kd fused to b2-microglobulin expressed in the baculovirus/insect cell system
Purification andLigand Bindingof a Soluble Class I Major Histocompatibility Complex MoleculeConsisting of the First Three Domains of H-2KdFused to,&-Microglobulin Expressed in the Baculovirus-Insect Cell System*
Summary
The foreign gene is cloned downstream of a strong very latepromoter and introduced via homologous recombination back into thegenome of Autographa californica nuclear polyhedrosis virus (AcNPV) (Miller, 1988;Luckow and Summers, 1989; Maeda, 1989). We describe here some characteristics of this purified soluble protein and demonstrate that itsinteraction column extensivsly, 1 liter of medium conditioned by AcNPV-SCKd-infected Sf9 cells was passed overthe column during a 6-h period, after which the column was washed with 20 column volumes of phosphate-buffered saline. Five to seven ing whole cells, infected Sf9 cells were harvested after 48 h, washed days after transfection, thveiral progenycontaining a mixture of wild in phosphate-buffered saline, resuspended X(2lo6cells/ml) in serumtype and recombinantviruses was submitted toa purification proce- free TC 100 medium in the presenceof [1s51]IASA-YIPSEAK(biotin)I dure based on limiting dilution in 96-well microtiter plates followed (1X lo cpm) for 6 h at 27 "C, and UV-irradiatedat 4 "C for 10 min by hybridization to a H-2Kdprobe.
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