Engineered phenylalanine ammonia lyase polypeptides

Patent metadata

Field	Value
Publication number	US-11401515-B2
Application number	US-202016831698-A
Country	US
Kind code	B2
Filing date	Mar 26, 2020
Priority date	Apr 18, 2013
Publication date	Aug 2, 2022
Grant date	Aug 2, 2022

Abstract

The present invention provides engineered phenylalanine ammonia-lyase (PAL) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered phenylalanine ammonia-lyase (PAL) polypeptides.

First claim

What is claimed is:

1. An engineered polynucleotide encoding an engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to reference sequence SEQ ID NO:4, wherein said amino acid sequence comprises an amino acid residue selected from alanine, aspartic acid, and glutamine at position 308, wherein said positions in said amino acid sequence are in reference to SEQ ID NO:4.

2. The engineered polynucleotide encoding the engineered polypeptide of claim 1 , wherein said engineered polypeptide exhibits an improved property selected from reduced sensitivity to proteolysis and increased tolerance to acidic pH, or a combination thereof, as compared to the reference sequence SEQ ID NO:4.

3. The engineered polynucleotide encoding the engineered polypeptide of claim 2 , wherein the improved property is selected from reduced sensitivity to proteolysis and/or increased tolerance to acidic pH.

4. The engineered polynucleotide encoding the engineered polypeptide of claim 1 , wherein said engineered polypeptide is resistant to proteolysis and acid stable.

5. The engineered polynucleotide encoding the engineered polypeptide of claim 4 , wherein said engineered polypeptide is resistant to proteolysis by at least one digestive tract enzyme, wherein said engineered polypeptide is resistant to proteolysis by chymotrypsin, trypsin, carboxypeptidases, and/or elastases.

6. The engineered polynucleotide encoding the engineered polypeptide of claim 5 , wherein said engineered polypeptide is deimmunized.

7. The engineered polynucleotide encoding the engineered polypeptide of claim 1 , wherein said polypeptide is purified.

8. The engineered polynucleotide sequence of claim 1 , wherein said polynucleotide sequence is operably linked to a control sequence.

9. The engineered polynucleotide sequence of claim 1 , wherein said polynucleotide sequence is codon-optimized.

10. An expression vector comprising at least one polynucleotide sequence of claim 1 , and at least one control sequence.

11. An expression vector comprising at least one polynucleotide sequence of claim 9 , and at least one control sequence.

12. The expression vector of claim 10 , wherein said control sequence is a promoter.

13. The expression vector of claim 11 , wherein said control sequence is a promoter.

14. A host cell transformed with the expression vector of claim 10 .

15. A host cell transformed with the expression vector of claim 11 .

16. A host cell transformed with the expression vector of claim 12 .

17. A host cell transformed with the expression vector of claim 13 .

18. A method of producing an engineered polypeptide in a host cell comprising culturing the host cell of claim 14 , under suitable culture conditions, such that at least one engineered polypeptide is produced.

19. A method of producing an engineered polypeptide in a host cell comprising culturing the host cell of claim 15 , under suitable culture conditions, such that at least one engineered polypeptide is produced.

20. A method of producing an engineered polypeptide in a host cell comprising culturing the host cell of claim 16 , under suitable culture conditions, such that at least one engineered polypeptide is produced.

21. A method of producing an engineered polypeptide in a host cell comprising culturing the host cell of claim 17 , under suitable culture conditions, such that at least one engineered polypeptide is produced.

22. The method of claim 18 , further comprising recovering at least one engineered polypeptide from the culture and/or host cells.

23. The method of claim 19 , further comprising recovering at least one engineered polypeptide from the culture and/or host cells.

24. The method of claim 20 , further comprising recovering at least one engineered polypeptide from the culture and/or host cells.

25. The method of claim 21 , further comprising recovering at least one engineered polypeptide from the culture and/or host cells.

26. The method of claim 22 , further comprising the step of purifying said at least one engineered polypeptide.

27. The method of claim 23 , further comprising the step of purifying said at least one engineered polypeptide.

28. The method of claim 24 , further comprising the step of purifying said at least one engineered polypeptide.

29. The method of claim 25 , further comprising the step of purifying said at least one engineered polypeptide.

30. A composition comprising at least one engineered polynucleotide of claim 1 .

31. The engineered polynucleotide of claim 1 , wherein said polynucleotide is purified.

Assignees

• Codexis Inc

Inventors

• Huisman Gjalt W
• Agard Nicholas J
• Mijts Benjamin
• Vroom Jonathan
• Zhang Xiyun

Classifications

• A61P3/00

  Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00) · CPC title

• A61P25/00

  Drugs for disorders of the nervous system · CPC title

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12Y403/01005

  Phenylalanine ammonia-lyase (4.3.1.5) (C12Y403/01023 - C12Y403/01025 takes precedence) · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title