Method for amidating polypeptides with basic amino acid C-terminals by means of specific endoproteases
The invention relates to a method for producing C-terminal amidated dibasic or polybasic peptides, consisting in reacting two peptides in the presence of trypsin biologically active enzymes and, if necessary, in purifying the thus obtainable compounds of formula (I) by means of protein chemistry.
1. A process for preparing a C-terminally amidated peptide of the formula (AA)-X—NH 2 wherein (AA) is a peptide that comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4 and wherein X is lysine or arginine; the process comprising reacting a peptide having the amino acid sequence of SEQ ID NO:7 with
(i) H-Arg-NH 2 in a 585:1 molar ratio in the presence of an enzyme having the biological activity of trypsin to produce the C-terminally amidated peptide of SEQ ID NO:4, or
(ii) H-Lys-NH 2 in a 144:1 molar ratio in the presence of an enzyme having the biological activity of trypsin to produce the C-terminally amidated peptide of SEQ ID NO:1,
and the resulting C-terminally amidated peptide is subjected to protein chemical purification.
2. The process as claimed in claim 1 , further comprising
a) expressing a fusion peptide which comprises a peptide having the sequence SEQ ID NO:7 with a recombinant host cell;
b) liberating the peptide having the sequence of SEQ ID NO:7 from said fusion peptide by enzymatic cleavage; and
c) purifying the liberated peptide from step b) and then reacting the purified peptide with H-Arg-NH 2 or H-Lys-NH 2 in the presence of an enzyme having the biological activity of trypsin; and
d) subjecting the resulting peptide to protein chemical purification and isolation.
3. The process as claimed in claim 2 , in which the liberation from the fusion protein takes place by enzymatic cleavage with enterokinase, factor Xa, Genenase, thrombin or trypsin.
4. The process as claimed in claim 3 , in which the fusion protein is expressed in a host/vector expression system selected from the group comprising E. coil, S. carnosus, Salmonella, Bacillus subtilis, Pseudomonas fluorescens, K. lactis, P. pastoris, Schizosaccharomyces pombe and S. cerevisiae.
5. A medicament comprising the C-terminally amidated peptide of SEQ ID NO:4.