INTEIN-MODIFIED ENZYMES, THEIR PRODUCTION AND INDUSTRIAL APPLICATIONS

• US 20130007919A1
• Filed: 11/05/2010
• Published: 01/03/2013
• Est. Priority Date: 11/06/2009
• Status: Active Grant

• Current Assignee: Agrivida, Inc. (Nippon Soda Company Limited)
• Original Assignee: Agrivida, Inc. (Nippon Soda Company Limited)
• Inventors: Lazar, Gabor, de la Vega, Humberto, Lessard, Philip, Raab, R. Michael, Apgar, James, Shen, Binzhang
• Granted Patent: US 10,407,742 B2
• Time in Patent Office: Days
• US Class Current: 800/298
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8246 Non-starch polysaccharides, e.g. cellulose, fructans, levans
• CPC Class Codes: C12N 15/8257 for the production of primary gene products, e.g. pharmaceutical products, interferon
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12N 9/248 Xylanases
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01008 Endo-1,4-beta-xylanase (3.2.1.8)

Abstract

Intein modified proteins, isolated nucleic acids encoding intein modified proteins, fragments of intein modified proteins, isolated nucleic acids encoding fragments of intein modified proteins, transgenic plants containing any of the foregoing, and antibodies recognizing epitopes on intein modified proteins are provided

Claims

1. An isolated protein having an amino acid sequence with at least 90% identity to a sequence selected from the group consisting of SEQ ID NOS:

2059-2089.

2. The isolated protein of claim 1, wherein the amino acid sequence has at least 95% identity to the sequence selected from the group consisting of SEQ ID NOS:

2059-2089.

3. The isolated protein of claim 1, wherein the amino acid sequence is the sequence selected from the group consisting of SEQ ID NOS:

2059-2089.

4. An isolated nucleic acid having a nucleotide sequence encoding an amino acid sequence with at least 90% identity to a sequence selected from the group consisting of SEQ ID NOS:

2059-2089.

5. The isolated nucleic acid of claim 4, wherein the amino acid sequence has at least 95% identity to the sequence selected from the group consisting of SEQ ID NOS:

2059-2089.

6. The isolated nucleic acid of claim 4, wherein the amino acid sequence is the sequence selected from the group consisting of SEQ ID NOS:

2059-2089.

7. A transgenic plant comprising an isolated protein having an amino acid sequence with at least 90% identity to a sequence selected from the group consisting of SEQ ID NOS:

2059-2089.

8. The transgenic plant of claim 7, wherein the amino acid sequence has at least 95% identity to the sequence selected from the group consisting of SEQ ID NOS:

2059-2089.

9. The transgenic plant of claim 7, wherein the amino acid sequence is the sequence selected from the group consisting of SEQ ID NOS:

2059-2089.

10. A transgenic plant comprising an isolated nucleic acid having a nucleotide sequence encoding an amino acid sequence with at least 90% identity to a sequence selected from the group consisting of SEQ ID NOS:

2059-2089.

11. The transgenic plant of claim 10, wherein the amino acid sequence has at least 95% identity to the sequence selected from the group consisting of SEQ ID NOS:

2059-2089.

12. The transgenic plant of claim 10, wherein the amino acid sequence is the sequence selected from the group consisting of SEQ ID NOS:

2059-2089.

13. The transgenic plant of claim 10, wherein the isolated nucleic acid further includes an expression construct.

14. The transgenic plant of claim 13, wherein the expression construct has the sequence of SEQ ID NO:

1 and the isolated nucleic acid is cloned into the expression construct by replacing the SEQ ID NO;

1 sequence that would be liberated by KpnI/EcoRI digestion of the expression construct between the KpnI site at nucleotides 9945-9950 of SEQ ID NO;

1 and the EcoRI site at nucleotides 12311-12315 of SEQ ID NO;

1 with the isolated nucleic acid with matching KpnI/EcoRI ends.

15. An isolated amino acid sequence comprising a contiguous amino acid sequence having at least 90% identity to 6, 10 to 50, 10 to 100, 10 to 150, 10 to 300, 10 to 400, 10 to 500, 10 to 600 or 10 to 658 contiguous amino acid residues of a protein having the sequence of any one of SEQ ID NOS:

2059-2089, wherein the protein has an intein sequence, an enzyme sequence, an upstream intein-extein junction, a downstream intein-extein junction and at least one amino acid change relative to at least one of SEQ ID NO;

111 or SEQ ID NO;

91, and the isolated amino acid sequence includes at least one of the upstream intein-extein junction, the downstream intein-extein junction or one or more of the at least one amino acid change relative to at least one of SEQ ID NO;

111 or SEQ ID NO;

91.

16. The isolated amino acid sequence of claim 15, wherein the contiguous amino acid sequence has 100% identity to the 6, 10 to 50, 10 to 100, 10 to 150, 10 to 300, 10 to 400, 10 to 500, 10 to 600 or 10 to 658 contiguous amino acid residues of a protein having the sequence of any of SEQ ID NOS:

2059-2089.

17. An antibody recognizing an epitope on the isolated amino acid sequence of claim 15.

18. An isolated nucleic acid having a sequence encoding a contiguous amino acid sequence having at least 90% identity to 6, 10 to 50, 10 to 100, 10 to 150, 10 to 300, 10 to 400, 10 to 500, 10 to 600 or 10 to 658 contiguous amino acid residues of a protein having the sequence of any one of SEQ ID NOS:

2059-2089, wherein the protein has an intein sequence, an enzyme sequence, an upstream intein-extein junction, a downstream intein-extein junction and at least one amino acid change relative to at least one of SEQ ID NO;

111 or SEQ ID NO;

91, and the isolated nucleic acid encodes at least one of the upstream intein-extein junction, the downstream intein junction or one or more of the at least one amino acid change relative to at least one of SEQ ID NO;

111 or SEQ ID NO;

91.

19. The isolated nucleic acid of claim 18, wherein the contiguous amino acid sequence has at least 95% identity to the 6, 10 to 50, 10 to 100, 10 to 150, 10 to 300, 10 to 400, 10 to 500, 10 to 600 or 10 to 658 contiguous amino acid residues of a protein having the sequence of any one of SEQ ID NOS:

2059-2089.

20. The isolated nucleic acid of claim 18, wherein the contiguous amino acid sequence has 100% identity to the 6, 10 to 50, 10 to 100, 10 to 150, 10 to 300, 10 to 400, 10 to 500, 10 to 600 or 10 to 658 contiguous amino acid residues of a protein having the sequence of any one of SEQ ID NOS:

2059-2089.

21-53. -53. (canceled)