Alpha amidating enzymes are thus useful in the conversion of recombinant precursor peptides to mature peptides.
US5789234 describes the production of an alpha-amidating enzyme by recombinant DNA techniques.
WO90/08194 relates to a process for production of C-terminal alpha amidated peptide from a precursor peptide by the use of a eukaryotic C terminal alpha amidating enzyme.
The invention also provides an enzyming having having peptidyl-a-hydroxyglycine alpha- amidating lyase activity characterised in that it can be produced by a method comprising the steps of: (i) cultivating a recombinant an Escherichia coii strain host cell comprising a nucleic acid construct comprising a nucleotide sequence encoding the enzyme, under conditions suitable for the expression of the enzyme; and (ii) recovering the enzyme from the super- natant after host cell disruption and centrifugation. T7 promoter region, Ampecillin resistance gene, lacl repressor region and origin of replication site are also shown in the vector map.
Also provided are enzymes capable of catalysing the conversion of a a-hydroxyglycine to an a-amide in a peptide, wherein said enzyme has an amino acid sequence comprising the following motif (named motif 1 ): Xaai Val Xaa are not Cys. Figure 2: SDS-PAGE showing the expression profile of RL9_THEMA TAP tagged Erythrobacter PAL-like domain (A) and rat PAL (B). control), Ind: After induction with 0.5 m M IPTG for 3 hours at 30°C, Sup: soluble fraction, Pel: Insoluble fraction.
Therefore a C-terminal alpha amide must be introduced in the recombinantly expressed peptides using for an example an ex vivo modification with alpha amidating enzymes.
Enzymatic modification of peptide precursors with a C-terminal Gly to a-amide by means of a bifunctional peptidyl a-amidating monooxygenase (PAM) is found in several eukaryotic organisms.
Symlin®, pramlintide acetate, which is an analogue of human amylin).
Human amylin is a 37 amino acid residue peptide which can be used for treating or preventing obesity and/or diabetes.
EP0448513 describes a process for recombinant expression of a peptidylglycine alpha- hydroxylating monooxygenase derived from Xenopus Laevis, comprising culturing insect cells transfected with a recombinant baculovirus to which a DNA coding for the peptidylglycine alpha-hydroxylating monooxygenase has been incorporated to produce the enzyme.
EP0465404 describes an enzyme (PHL; PAL) derived from Xenopus Laevis catalysing the cleavage of the N-C bond in the a-hydroxylglycine moiety of a C-terminally a- hydroxylated peptide, the cloning of the enzyme and its recombinant expression in insect cells.
Accordingly, the C-terminal of amylin needs to be amidated in order to obtain full biological activity.