Both eukaryotic and prokaryotic expression vectors are provided having a transcriptional promoter followed downstream by a DNA sequence which encodes amidating enzyme.
The vector selected is one capable of directing the expression of polypeptides in the host selected, and preferred hosts are transected with the described vectors.
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In vertebrates, peptidylglycine alpha-amidating monooxygenase (PAM) is a multifunctional protein found in secretory granules.
Although PAM is encoded by a single gene, soluble and membrane-bound monofunctional and bifunctional forms are generated by tissue-specific alternative splicing and endoproteolytic cleavage.
The first step of the reaction is catalysed by peptidylglycine alpha-hydroxylating monooxygenase (PHM), and is dependent on copper, ascorbate and molecular oxygen; peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyses the second step of the reaction [PMID: 1448112].We purified an alpha-amidating enzyme from equine serum by simplified steps including substrate affinity chromatography.With the purified enzyme, we detected an intermediate of the alpha-amidating reaction by high performance liquid chromatography analysis.Drosophila PHM encodes an active enzyme that is required for peptide amidation in vivo [PMID: 10993678], while both PAL proteins display PAL enzymatic activity and are involved in neuroendocrine biosynthesis [PMID: 15198673].The peptidylglycine alpha-amidating enzyme catalyzes a reaction that transforms a carboxyl-terminal glycine-extended precursor into a carboxyl-terminal alpha-amidated peptide.
PHM share protein sequence similarity with dopamine-beta-monooxygenases (DBH), a class of ascorbate-dependent enzymes that requires copper as a cofactor and uses ascorbate as an electron donor.