Intein-modified enzymes, their production and industrial applications
First Claim
Patent Images
1. An intein modified protein comprising a target protein and an intein inserted into the target protein at a site i) immediately prior to a cysteine, threonine, or serine residue of the target protein, ii) within ten angstroms from an active site of the target protein, and iii) within 10 amino acids of a loop-β
- -sheet junction or within 10 amino acids of a loop-α
-helix junction of the target protein, wherein the target protein is a xylanase or a cellulase.
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Accused Products
Abstract
A method of predicting an intein insertion site in a protein that will lead to a switching phenotype is provided. The method includes identifying a plurality of C/T/S sites within the protein; selecting from the plurality of C/T/S/ sites those that are ranked 0.75 or higher by a support vector machine, within ten angstroms of the active site of the protein, and at or near a loop-β-sheet junction or a loop-α-helix junction. A method of controlling protein activity and hosts including proteins with controlled activity are also provided. Also, intein modified proteins and plants containing intein modified proteins are provided.
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Citations
33 Claims
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1. An intein modified protein comprising a target protein and an intein inserted into the target protein at a site i) immediately prior to a cysteine, threonine, or serine residue of the target protein, ii) within ten angstroms from an active site of the target protein, and iii) within 10 amino acids of a loop-β
- -sheet junction or within 10 amino acids of a loop-α
-helix junction of the target protein, wherein the target protein is a xylanase or a cellulase. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
- -sheet junction or within 10 amino acids of a loop-α
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12. An intein modified protein having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:
- 1692-1695, 1698, 1719, 1732, 1739-1742, 1745-1750, 1753-1766, 1768-1769, 1772-1773.
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13. A transgenic plant including an expression construct encoding an intein modified protein comprising a target protein and an intein inserted into the target protein at a site i) immediately prior to a cysteine, threonine, or serine residue of the target protein, ii) within ten angstroms from an active site of the target protein, and iii) within 10 amino acids of a loop-α
- -sheet junction or within 10 amino acids of a loop-β
-helix junction of the target protein, wherein the target protein is a xylanase or a cellulase. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
- -sheet junction or within 10 amino acids of a loop-β
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24. A transgenic plant including an expression construct encoding an intein modified protein having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:
- 1692-1695, 1698, 1719, 1732, 1739-1742, 1745-1750, 1753-1766, 1768-1769, 1772-1773.
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25. A method of making an intein modified protein including:
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(a) identifying a plurality of cysteine, serine or threonine sites within the protein, whether native or introduced, and (b) carrying out at least one of steps (i) to (iv) (i) analyzing the local sequence of each of the plurality of cysteine, serine or threonine sites with a support vector machine; (ii) identifying an active site of the protein, wherein a respective distance separates the active site and each of the plurality of cysteine, serine or threonine sites, and identifying the respective distance between the active site and each of the plurality of cysteine, serine or threonine sites; and (iii) identifying a secondary structure of the protein at each of the plurality of cysteine, serine or threonine sites; the method further including (c) inserting an intein into the protein at a site immediately before at least one of the cysteine, serine or threonine sites that is in a position selected from at least one of the group consisting of i) one of the plurality of cysteine, serine or threonine sites ranked 0.75 or higher by the support vector machine, ii) one of the plurality of cysteine, serine or threonine sites within ten angstroms of the active site and iii) one of the plurality of cysteine, serine or threonine sites within ten amino acids from a loop-β
-sheet junction or one of the plurality of cysteine, serine or threonine sites within ten amino acids from a loop-α
-helix junction. - View Dependent Claims (26)
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- 27. The method of claim 27 method further comprising screening the intein modified protein to determine if the intein modified protein has a permissive or switching phenotype.
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30. An expression construct comprising a nucleic acid having a sequence with at least 90% identity to the sequence of SEQ ID NO:
- 1.
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31. The expression construct of claim 31, wherein the nucleic acid has 100% identity to the sequence of SEQ ID NO:
- 1.
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32. A mutant intein comprising an amino acid sequence having 90% identity to a sequence selected from the group consisting of SEQ ID NOS:
- SEQ ID NOS;
92-103, 1675, 1678-1681, 1689, 1691, 1700-1708, and 1710. - View Dependent Claims (33)
- SEQ ID NOS;
Specification
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Current AssigneeAgrivida, Inc. (Nippon Soda Company Limited)
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Original AssigneeAgrivida, Inc. (Nippon Soda Company Limited)
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InventorsLessard, Philip, Lazar, Gabor, de la Vega, Humberto, Apgar, James, Shen, Binzhang, Raab, R. Michael
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Granted Patent
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Time in Patent OfficeDays
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Field of Search
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US Class Current435/18
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CPC Class CodesC12N 15/8216 Methods for controlling, re...C12N 15/8241 Phenotypically and genetica...C12N 15/8246 Non-starch polysaccharides,...C12N 9/2437 Cellulases (3.2.1.4; 3.2.1....C12N 9/248 XylanasesC12N 9/2482 Endo-1,4-beta-xylanase (3.2...C12Y 302/01004 Cellulase (3.2.1.4), i.e. e...C12Y 302/01008 Endo-1,4-beta-xylanase (3.2...
