A SOLUBLE, SINGLE-CHAIN KD MOLECULE PRODUCED BY YEAST SELECTS A PEPTIDE REPERTOIRE INDISTINGUISHABLE FROM THAT OF CELL-SURFACE-ASSOCIATED KD

Peptide binding to a soluble, single-chain K(d) protein produced by the yeast strain Kluyveromvces lactis, and to K(d) molecules on K(d)-expressing cells (P815) was studied using radiolabeled K(d)-restricted peptides. The stability of the peptide-K(d) complexes formed was monitored in the absence and presence of unlabeled competitor peptides. Radioiodination of the Tyr anchor residue in position 2 of the peptide interferes with binding. A K(d)-biased peptide library and a modified antigenic peptide in which a second Tyr was added in positions 6 and 8, respectively, were therefore used to assay binding. Recombinant and cell-associated K(d) Molecules are very similar in the following respects: the ease with which the proteins can be loaded with labeled peptide; the spectrum of peptides selected from a peptide library; the stability of the labeled peptide-K(d) complex formed; and the ability to partially dissociate the class I-peptide complex with exogenous, unlabeled peptides. These results imply that measurements of peptide binding to soluble K(d) molecules are a reliable indicator of the peptide-binding properties of K(d) proteins on living cells. The large quantities of soluble recombinant K(d) protein currently available represent an invaluable tool not only for dissecting the molecular mechanisms of antigen presentation but also for vaccinations and the design of T cell-specific toxins.