Intein-modified enzymes, their production and industrial applications

• US 9,464,333 B2
• Filed: 11/05/2010
• Issued: 10/11/2016
• 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: Apgar, James, Lessard, Philip, Raab, R. Michael, Shen, Binzhang, Lazar, Gabor, de la Vega, Humberto
• Primary Examiner(s): DEVEAU ROSEN, JASON
• Application Number:
  US13/508,234
• Publication Number:
  US 20130036517A1
• Time in Patent Office: 2,167 Days
• Field of Search: None
• US Class Current: 1/1
• 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 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

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.

Claims

1. An intein modified protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:

2374, 2376-2378, 2383, 2385-2386, 2410, 2412-2413, 2420, 2422-2425, 2428, 2430-2431, 2436, 2442-2444, 2451, 2454-2458, 2460-2461, 2465-2468, 2471-2474, 2477-2480, 2482-2483, 2493-2494, 2500-2501, 2504, 2507-2513, 2517-2519, 2530-2531, 2533-2537, 2539-2543, 2545, 2548-2549, 2555-2557, 2559, 2565, 2569-2571, 2573-2575, 2579-2582, 2584-2597, 2600, 2602-2605, 2607-2621, 2624-2626, 2629-2634, 2636-2639, 2643, 2645-2648, 2650, 2652, 2656-2658, 2661, 2664, 2666-2672, 2674, 2677-2683, 2685, and 3315-3322.

6. The intein modified protein of claim 1 comprising the amino acid sequence of SEQ ID NO:

3315.

2. A nucleic acid comprising a nucleotide sequence encoding an intein modified protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:

2374, 2376-2378, 2383, 2385-2386, 2410, 2412-2413, 2420, 2422-2425, 2428, 2430-2431, 2436, 2442-2444, 2451, 2454-2458, 2460-2461, 2465-2468, 2471-2474, 2477-2480, 2482-2483, 2493-2494, 2500-2501, 2504, 2507-2513, 2517-2519, 2530-2531, 2533-2537, 2539-2543, 2545, 2548-2549, 2555-2557, 2559, 2565, 2569-2571, 2573-2575, 2579-2582, 2584-2597, 2600, 2602-2605, 2607-2621, 2624-2626, 2629-2634, 2636-2639, 2643, 2645-2648, 2650, 2652, 2656-2658, 2661, 2664, 2666-2672, 2674, 2677-2683, 2685, and 3315-3322.

3. A transgenic plant comprising the nucleic acid of claim 2.

4. The transgenic plant of claim 3 further comprising an expression construct, wherein the nucleic acid is contained within the expression construct.

5. The transgenic plant of claim 4, wherein the expression construct comprises the sequence of SEQ ID NO:

1.