Novel peptidyl alpha-hydroxyglycine alpha-amidating lyases
AssigneeNovo Nordisk A/S
InventorAllan Christian Shaw
Life of this patent
Estimating the term…
Abstract
The present patent application concerns an enzyme capable of catalysing the conversion of a α-hydroxyglycine to an α-amide and the use of such enzymes for producing a C-terminal α-amidated peptide.
Claims13
An isolated polypeptide capable of catalysing the conversion of a α-hydroxyglycine to an α-amide, wherein said polypeptide comprises an amino acid sequence comprising the following motif 1:
- Xaa1 Val Xaa2 Asp Arg Xaa3 Xaa4 Xaa5 Arg Xaa6 Gln Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Trp;
- where Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9, Xaa10, Xaa11 Xaa12, Xaa13. Xaa14, Xaa15, and Xaa16 are selected independently from a natural accurring amino acid, provided that Xaa1 and Xaa7 are not Cys.
An isolated polypeptide capable of catalysing the conversion of a α-hydroxyglycine to an α-amide, which polypeptide is selected from the group consisting of:
- (a) a polypeptide comprising an amino acid sequence having at least 80% identity to amino acids 2-306 of SEQ ID No. 1;
- (b) a polypeptide comprising an amino acid sequence having at least 80% identity to amino acids 3-336 of SEQ ID No. 2;
- (c) a polypeptide comprising an amino acid sequence having at least 80% identity to amino acids 3-305 of SEQ ID No. 3;
- (d) a polypeptide comprising an amino acid sequence having at least 80% identity to amino acids 3-279 of SEQ ID No. 4;
- and
- (f) a polypeptide having peptidyl-α-hydroxyglycine alpha-amidating lyase activity characterised in that it has an amino acid sequence comprising no cysteine residues, or at most 1 or at most 2 cysteine residues.
An isolated polypeptide having peptidyl-α-hydroxyglycine alpha-amidating lyase activity characterised in that it can be expressed as a soluble protein in E. coli.
A method for producing an enzyme with peptidyl-α-hydroxyglycine alpha-amidating lyase (PAL) activity comprising the steps of:
- (i) cultivating a recombinant expression host cell of non-mammalian origin, which comprises 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
- (a) the supernatant after cell disruption and centrifugation and/or
- (b) the growth media
- wherein the host cell is a of non-mammalian origin, e.g. an Escherichia coli strain, and wherein said enzyme is soluble when recovered in step (ii).
Dependencies are inferred from the claim text.