[Primary structure of human class II histocompatibility antigens (HLA-D). I. Isolation, purification and characterization of the HLA-D alpha/beta chain complex from a homozygous lymphoblastoid B cell line, H2LCL (HLA-A3,3;B7,7;Dw2, 2;DR2,2;MT1,1;DC1,1;MB1,1].
The complex of alpha and beta chains of HLA-D membrane antigens has been isolated from a lymphoblastoid homozygous B cell line, H2LCL (HLA-A3,3; B7,7; Dw2,2; DR2,2; MT1,1; DC1,1; MB1,1), by an exclusively chemical two-step procedure and characterized by electrophoresis as well as isoelectric focusing in polyacrylamide gel. Cells were gained using long term cultivation in large scale, the crude membrane by differential centrifugation. The proteins of the crude membrane were then solubilized in NP-40, pH 5.0. The first purification step for HLA-D antigens consisted in an ion-exchange chromatography on carboxymethyl cellulose using the solubilization buffer. By this procedure the complex of proteins with relative molecular masses of Mr approximately 34 000 and Mr approximately 29 000 was in a high percentage not bound to the carboxymethyl cellulose. The bound fraction contained the HLA-A, -B and -C antigens and a component with Mr approximately 31 000 corresponding to the well known Ii-fraction. The bound proteins could be recovered from the column by a sodium chloride gradient. The proteins not bound to the carboxymethyl cellulose were precipitated with acetone, dissolved, dialysed against SDS buffer, pH 7.2 and then submitted to the second purification step, the Sephacryl S-300 chromatography. By this procedure the corresponding complex could be further separated from higher and lower molecular proteins. The complex was used as the starting material for the separation of alpha and beta chains. Amino-acid sequences established of the isolated chains have already been communicated.
D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange / Q000379:methods; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D000949:Histocompatibility Antigens Class II / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D006720:Homozygote; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods
D000949:Histocompatibility Antigens Class II
Primärstruktur der menschlichen Histokompatibilitätsantigene der Klasse II (HLA-D) I. Isolierung, Reinigung und Charakterisierung des alpha/beta-Ketten-Komplexes des HLA-D einer homozygoten, lymphoblastoiden B-Zell-Linie, H2LCL (HLA-A3,3; B7,7; Dw2,2; DR2,2; MT1,1; DC1,1; MB1.1).