Because the a1(II) chains contain a relatively small number of methionyl residues (e.g., 10 residues per a chain in bovine CII), cyanogen bromide (CNBr) cleavage of bovine a1(II) yields a mixture of eleven peptides. By convention, the CNBr peptides were named according to their order of elution from the ion-exchange column (Fig. 15.5.1). The peptides range in size from dipeptide to peptides of 345 residues (Table 15.5.2). For studies of CIA in mice, the peptides CB11, CB8, and CB10 are of particular interest. For H-2q mice (DBA/1LacJ), CB11 has been shown to contain the dominant T and B cell epitopes sufficient to induce arthritis (Terato et al, 1985; Brand et al., 1994). For H-2r mice, CB10 has been shown to contain the dominant T cell epitope and the most effective tolerogenic determinant, whereas CB8 has been shown to contain arthritogenic T and B cell epitopes (Myers et al., 1995). Although HPLC is the most powerful technique in the purification of the major CNBr peptides, it has proven difficult to scale up the purification process to yield sufficient quantities of peptides necessary for experimentation. The following describes procedures for preparation of milligram quantities of CNBr peptides using a combination of ion-exchange and size-exclusion chromatography.
Figure 15.5.1 Purification of cyanogen bromide fragments of chick CII by ion-exchange chromatography.
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