Engineered phenylalanine ammonia lyase polypeptides

Patent metadata

Field	Value
Publication number	US-10358639-B2
Application number	US-201816166359-A
Country	US
Kind code	B2
Filing date	Oct 22, 2018
Priority date	Apr 18, 2013
Publication date	Jul 23, 2019
Grant date	Jul 23, 2019

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 a 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 a glycine at position 290, and wherein the positions in said amino acid sequence are in reference to SEQ ID NO:4.

2. The engineered polynucleotide of claim 1 , wherein said encoded engineered polypeptide comprises at least one or more of the following substitutions or substitution sets selected from 39/91/158/180/195/256/290/399/459/463, 39/91/158/180/290/394/399/474/522/524, 39/91/256/290/394/399/404/407/522/524, 91/158/243/256/290/399/407/459/463/474/522/524, and 256/290/404/407/474/522, wherein said positions in said amino acid sequence are in reference to SEQ ID NO:4.

3. The engineered polynucleotide of claim 1 , wherein said encoded engineered polypeptide comprises at least one or more of the following substitutions or substitution sets selected from 39/91/256/290/307/399/404/407/522/524, 39/91/256/290/307/404/407/524, 39/91/256/290/399/404/407/522, 39/91/290/307/407, 39/91/290/307/407/524, and 39/256/290/307/404/407, wherein said positions in said amino acid sequence are in reference to SEQ ID NO:4.

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

5. The engineered polynucleotide encoding an engineered polypeptide of claim 4 , wherein the improved property is selected from reduced sensitivity to proteolysis and/or increased tolerance to acidic pH.

6. The engineered polynucleotide encoding an engineered polypeptide of claim 4 , wherein said engineered polypeptide is resistant to proteolysis, acid stable, and/or deimmunized.

7. The engineered polynucleotide encoding an engineered polypeptide of claim 5 , 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.

8. The engineered polynucleotide encoding an engineered polypeptide of claim 6 , wherein said engineered polypeptide is deimmunized.

9. The engineered polynucleotide encoding an engineered polypeptide of claim 1 , wherein said polypeptide is purified.

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

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

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

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

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

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

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 host cell comprising the engineered polynucleotide of claim 1 .

19. 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.

20. 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.

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

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

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

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

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

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

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

Assignees

• Codexis Inc

Inventors

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

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• 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

• 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

• C12Y403/01024

  Phenylalanine ammonia-lyase (4.3.1.24) · CPC title