Andreas Meyerhans

Peptide motifs of HLA-A3, -A24, and -B7 molecules as determined by pool sequencing.

As a source of HLA molecules, the mouse mastocytoma P815 transfected with the respective HLA gene was used. P815-A3 and P815-B7 cells were a gift from J. Maryanski, P815-A24 a gift from E LemonrLier (Layet et al. 1984; Maryanski et al. 1986; Pala et al. 1988). Transfectants were expanded in tissue culture with Dulbeccos modified Eagle medium/high glucose supplemented with 25 mM HEPES, 10% FCS and antibiotics. Two times 10 l0 cells were pelleted and lysed with 1% NP40 in phosphate buffered saline. HLA molecules were precipitated, using solid phase bound PA2.6 antibodies (specific for HLA-A, -B, -C; Parham and Bodmer 1978) as described (Falk et al. 1991). Precipitates were treated with trifluoroacetic acid in order to dissociate peptides from MHC molecules. The supernatant, containing the peptides, was separated by reversed phase HPLC, using Pharmacia equipment (Pharmacia, Freiburg, Germany) and a 4.0 minx250 mm C2/C18 column. The fractions known from previous experience (Falk et al. 1991) to contain peptides were pooled and sequenced by Edman degradation, using either a model 477 or 476 protein sequencer (Applied Biosystems, Weiterstadt, Germany). Interpretation of pool sequencing data was done as described earlier (Falk