Nucleic acids encoding modified relaxin polypeptides
Modified relaxin polypeptides and their uses thereof are provided.
1. An isolated nucleic acid encoding a modified relaxin polypeptide chain comprising a non-naturally encoded amino acid, wherein: (a) said isolated nucleic acid comprises a selector codon that encodes said non-naturally encoded amino acid; (b) the encoded modified relaxin polypeptide comprises a relaxin A chain polypeptide having a sequence at least 95% identical to SEQ ID NO: 4; and (c) said non-naturally encoded amino acid occurs at residue 1 of the A chain of SEQ ID NO:4 or the corresponding position of said modified relaxin polypeptide, and said non-naturally encoded amino acid comprises a functional group selected from a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group.
2. The isolated nucleic acid of claim 1 , wherein said nucleic acid encodes a relaxin A chain polypeptide having the sequence of SEQ ID NO: 4 containing said non-naturally occurring amino acid.
3. The isolated nucleic acid of claim 2 , wherein said nucleic acid further encodes a relaxin B chain polypeptide of SEQ ID NO: 5 or SEQ ID NO: 6.
4. The isolated nucleic acid of claim 1 , wherein said nucleic acid further encodes a relaxin B chain polypeptide having a sequence at least 95% identical to SEQ ID NO: 5 or SEQ ID NO: 6.
5. The isolated nucleic acid of claim 1 , wherein the non-naturally encoded amino acid is a phenylalanine analog or derivative.
6. The isolated nucleic acid of claim 1 , wherein the non-naturally encoded amino acid has the structure:
wherein the R group is any substituent other than the side chain found in alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.
7. The isolated nucleic acid of claim 1 , wherein the non-naturally encoded amino acid comprises para-acetyl-L-phenylalanine or para-azido-L-phenylalanine.
8. A host cell comprising the isolated nucleic acid of claim 1 .
9. The host cell of claim 8 , wherein said host cell further comprises an orthogonal tRNA that recognizes said selector codon and inserts said non-naturally encoded amino acid during translation of said isolated nucleic acid.
10. The host cell of claim 9 , wherein said host cell further comprises an orthogonal tRNA synthetase that synthesizes said orthogonal tRNA.
11. The host cell of claim 9 , wherein said non-naturally encoded amino acid comprises para-acetyl-L-phenylalanine.
12. The isolated nucleic acid of claim 1 , wherein the non-naturally encoded amino acid is selected from a para-substituted, ortho-substituted, or meta-substituted phenylalanine comprising a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group.
13. An in vitro translation system comprising the isolated nucleic acid of claim 1 .
14. The in vitro translation system of claim 13 , wherein said in vitro translation system further comprises an orthogonal tRNA that recognizes said selector codon and inserts said non-naturally encoded amino acid during translation of said isolated nucleic acid.
15. The in vitro translation system of claim 14 , wherein said in vitro translation system further comprises an orthogonal tRNA synthetase that synthesizes said orthogonal tRNA.
16. The in vitro translation system of claim 14 , wherein said non-naturally encoded amino acid comprises para-acetyl-L-phenylalanine.
17. The isolated nucleic acid of claim 1 , wherein said selector codon comprises an amber codon, ochre codon, or opal codon.
18. The isolated nucleic acid of claim 1 , wherein said isolated nucleic acid further comprises a nucleic acid encoding a secretion signal sequence.
19. An isolated nucleic acid encoding a modified relaxin polypeptide chain comprising a non-naturally encoded amino acid, wherein: (a) said isolated nucleic acid comprises a selector codon that encodes said non-naturally encoded amino acid; (b) the encoded modified relaxin polypeptide comprises the relaxin A chain polypeptide of SEQ ID NO: 4 containing one non-naturally encoded amino acid substitution; and (c) said non-naturally encoded amino acid occurs at residue 1 of the A chain of SEQ ID NO:4 or the corresponding position of said modified relaxin polypeptide, and said non-naturally encoded amino acid comprises a para-acetyl-L-phenylalanine.