Recently, the sequence of mitochondrial chaperonin 10 from Rattus norvegicus (rat cpn10), with N-terminal acetylation, has been published. Two syntheses of rat cpn10 were performed, the first using a classical carbodiimide-mediated double coupling protocol (Method A) and the second a more efficient HBTU/HOBT/single coupling procedure (Method B). The latter also involved the application of a capping procedure, using N-(2-chlorobenzyloxycarbonyloxy)succinimide [Z(2-Cl)-OSu]. The crude protein from Method A was purified using a two-step isoelectric focusing/RP-HPLC scheme and found to contain a high proportion of a deletion peptide (less Gln60). Conversely, rat cpn10 from Method B was purified to homogeneity by one-step RP-HPLC, using a reversible lipophilic chromatographic probe. The proportion of biologically active heptameric structure was directly related to the purity of the protein and attained 84% with material from Method B. The addition of Ca/Mg ions, pH 7.2, or a heating/cooling cycle increased the proportion of heptamer for less pure protein. Shorter sequences were found not to fold into heptamers, suggesting that aggregation/folding motifs are located in 1-25 and 77-101 regions of rat cpn10. The heptameric cpn10 (Method B) bound correctly to GroEL from E. coli, demonstrating that N-terminal acetylation is not necessary for its folding and binding to bacterial cpn60.
|Original language||English (US)|
|Number of pages||6|
|Journal||Biomedical peptides, proteins & nucleic acids : structure, synthesis & biological activity|
|State||Published - Jan 1 1994|