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.
NOVEL PEPTIDYL ALPHA-HYDROXYGLYCINE ALPHA-AMIDATING LYASES FIELD OF THE INVENTION The present invention relates to isolated polypeptides having peptidyl-a-hydroxyglycine alpha-amidating lyase activity, methods for preparing such polypeptides and the use of such polypeptides in processes for producing C-terminal a-amidated peptides.BACKGROUND OF THE INVENTION In multicellular organisms certain peptides ("precursors"), like neuropeptides, are post- translationally modified in a series of enzymatic steps that cleave and further modify peptide substrates to yield fully functional bioreactive peptides.US 20060292672 describes a cell line for expressing PAM or one of its two catalytic domains.EP2172550 describes a recombinant C-terminal alpha-amidating enzyme derivative which lack the formation of at one of the five disulfide bonds normally occurring in a C- terminal alpha-amidating enzyme derived from Xenopus laevis and method of producing said derivative recombinantly in E.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.
Alpha amidating enzymes are thus useful in the conversion of recombinant precursor peptides to mature peptides.
US4708934 describes a peptidyl-glycine a-amidating monooxygenase extracted from medullary thyroid carcinoma cell lines and tissue samples.
Also described is a method for eukaryotic expression of these C terminal alpha amidating enzymes.
WO89/02460 describes a bovine derived PAM enzyme, its cloning, c DNA and expression by recombinant DNA technology.
Accordingly, the C-terminal of amylin needs to be amidated in order to obtain full biological activity.