Biochemical purification of detergent-solubilized H-2 alloantigens

A multistep Chromatographic fractionation scheme is described for purifying the H-2K^b and H-2D^b major histocompatibility antigens as isolated from the murine lymphoblastoid cell line EL4 (H-2^b haplotype). The membrane-integrated antigen molecules were solubilized with the non-ionic detergent NP-40 and were purified by gel filtration chromatography, ion exchange chromatography and affinity chromatography with lentil lectin conjugated to Sepharose. During the latter procedure, use of a linear gradient monosaccharide elution effected partial separation of the H-2K^b and H-2D^b antigens. At this stage the H-2 glycoproteins were highly purified based on several criteria. Upon polyacrylamide gel electrophoresis in SDS the major band migrates with a mol. wt of approximately 45,000 daltons corresponding to the mol. wt of antigens obtained by immunoprecipitation. Moreover, near identity of the profiles of the arginine-containing tryptic peptides from chromatographically-purified and immunoprecipitated H-2K^b preparations suggests that a high degree of homogeneity has been achieved in the Chromatographic purification. As is demonstrated, mg quantities of the H-2K^b and the H-2D^b antigens can be isolated and partially separated from each other by this purification scheme thereby opening the way for structural studies of the H-2K and H-2D molecules by a variety of biochemical methods.