Primärstruktur der menschlichen Histokompatibilitätsantigene der Klasse II (HLA-D). I. Isolierung, Reinigung und Charakterisierung des α/β-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)

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Abstract
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 2-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 1st 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 MW 34,000 and MW .apprx. 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 MW .apprx. 31,000 corresponding to the well known li-fraction. The bound proteins could be recovered from the column by a NaCl gradient. The proteins not bound to the carboxymethyl cellulose were precipitated with acetone, dissolved, dialysed against SDS [sodium dodecyl sulfate] 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.

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