Asx-specific protein ligases and uses thereof
The present invention lies in the technical field of enzyme technology and specifically relates to enzymes having Asx-specific ligase and cyclase activity and to nucleic acids encoding those as well as methods of the manufacture of said enzymes. The enzymes having Asx-specific ligase and cyclase are isolated from plants of the Violaceae family. Further encompassed are methods and uses of these enzymes.
1 . A recombinant polypeptide having protein ligase activity comprising:
(i) the amino acid sequence as set forth in SEQ ID NO: 1; or
(ii) an amino acid sequence of at least 90% identity with SEQ ID NO:1 and comprises:
(a) the amino acid residue W or Y at position 195, the amino acid residue I, C, A or V at position 196, and the amino acid residue T, A or V at position 197 of SEQ ID NO:1;
(b) the amino acid residue A or G at position 126 and, the amino acid A or P at position 127 of SEQ ID NO:1, but not GP at positions 126 and 127 of SEQ ID NO: 1; and
(c) at least two of the amino acid residues selected from: the amino acid residue N at position 19 of SEQ ID NO: 1; the amino acid residue H at position 124 of SEQ ID NO: 1; and the amino acid residue C at position 166 of SEQ ID NO:1.
2 . The recombinant polypeptide of claim 1 , wherein the recombinant polypeptide comprises:
(i) the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:3; or
(ii) an amino acid sequence of at least 90% identity with SEQ ID NO:2 or 3 and comprises:
(a) the amino acid residue W or Y at position 195, the amino acid residue I, C, A or V at position 196, and the amino acid residue T, A or V at position 197 of SEQ ID NO:1;
(b) the amino acid residue A or G at position 126 and, the amino acid residue A or P at position 127 of SEQ ID NO: 1, but not GP at positions 126 and 127 of SEQ ID NO: 1; and
(c) at least two of the amino acid residues selected from: the amino acid residue N at position 19 of SEQ ID NO: 1; the amino acid residue H at position 124 of SEQ ID NO: 1; and the amino acid residue C at position 166 of SEQ ID NO:1.
3 . The recombinant polypeptide of claim 1 , wherein said polypeptide comprises: (i) the amino acid residue A at position 126 and the amino acid A or P at position 127 of SEQ ID NO: 1,
(ii) the amino acid residue G at position 126 and the amino acid A at position 127 of SEQ ID NO:1,
(iii) the amino acid residue W or Y at position 195, the amino acid residue I or V at position 196, and the amino acid residue T, A or V at position 197 of SEQ ID NO: 1,
(iv) the amino acid residue W at position 195 of SEQ ID NO: 1, the amino acid residue I or V at position 196 of SEQ ID NO: 1, and the amino acid residue T at position 197 of SEQ ID NO:1,
(v) the amino acid residues R at position 21, H at position 22, D at position 123, E at position 164, S at position 194, and D at position 215 of SEQ ID NO:1,
(vi) the amino acid residues C at position 199 and 212 of SEQ ID NO:1,
or combinations thereof.
4 . A nucleic acid molecule encoding the polypeptide according to claim 1 .
5 . A vector comprising the nucleic acid molecule of claim 4 .
6 . The vector of claim 5 , wherein said vector further comprises regulatory elements for controlling expression of said nucleic acid molecule.
7 . A host cell comprising the nucleic acid molecule of claim 4 , wherein, optionally, the host cell is an insect cell.
8 . A method for cyclizing a peptide or for ligating at least two peptides, the method comprising incubating said peptide or said at least two peptides with the recombinant polypeptide of claim 1 under conditions that allow cyclization of said peptide or ligation of said peptides.
9 . A solid support material comprising the recombinant polypeptide according to claim 1 immobilized thereon.