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 systemJournal of Biological Chemistry
ORCiDDavid M. Ojcius: 0000-0003-1461-4495
AbstractA 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 SF18.104.22.168. 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.
Citation InformationFrancois Godeau, Immanuel F. Luescher, David M. Ojcius, Cecile Saucier, et al.. "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" Journal of Biological Chemistry Vol. 267 Iss. 34 (1992) p. 24223 - 24229 ISSN: 0021-9258
Available at: http://works.bepress.com/david-ojcius/190/