ES2538360T3 - Métodos para aumentar la secreción de polipéptidos que tienen actividad biológica - Google Patents

Métodos para aumentar la secreción de polipéptidos que tienen actividad biológica Download PDF

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ES2538360T3
ES2538360T3 ES07868319.0T ES07868319T ES2538360T3 ES 2538360 T3 ES2538360 T3 ES 2538360T3 ES 07868319 T ES07868319 T ES 07868319T ES 2538360 T3 ES2538360 T3 ES 2538360T3
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fusion protein
endoglucanase
cel45
gene
polynucleotide
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Sandra Merino
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Novozymes Inc
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    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
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    • C12P19/02—Monosaccharides
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion

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Abstract

Método para producir una enzima de beta-glucosidasa, que comprende: (a) transformación de una célula huésped fúngica con un constructo de proteína de fusión que codifica una proteína de fusión, donde el constructo de proteína de fusión comprende: (i) un primer polinucleótido que comprende una secuencia de nucleótidos que codifica un péptido señal obtenido a partir de un gen de endoglucanasa (cel45) de Humicola insolens CEL45; (ii) un segundo polinucleótido que comprende una secuencia de nucleótidos que codifica al menos un dominio catalítico de una endoglucanasa obtenida a partir de un gen de endoglucanasa (cel45) de Humicola insolens CEL45; y (iii) un tercer polinucleótido que comprende una secuencia de nucleótidos que codifica una enzima de betaglucosidasa obtenida a partir de Aspergillus oryzae, donde la proteína de fusión es SEC ID nº: 74, (b) cultivo de la célula huésped fúngica transformada bajo condiciones adecuadas para la producción de la proteína de fusión; y (c) recuperación de la proteína de fusión del medio de cultivo.

Description

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E07868319
02-06-2015
[0065] Secuencias de codificación de péptido señal eficaces para células huésped fúngicas filamentosas son la secuencia codificante del péptido señal obtenida a partir de la endoglucanasa de Humicola insolens.
[0066] En un aspecto preferido, la secuencia codificante del péptido señal se obtiene a partir de un gen de Humicola insolens que codifica una endoglucanasa V. En un aspecto más preferido, la secuencia codificante de péptido señal codifica los aminoácidos 1 a 21 de SEC ID nº: 2. En otro aspecto más preferido, la secuencia codificante del péptido señal es los nucleótidos 1 a 63 de SEC ID nº: 1.
[0067] La proteína de fusión puede comprender además un segundo péptido señal que se una con el componente de beta-glucosidasa de la proteína de fusión. La secuencia codificante del péptido señal puede ser la secuencia codificante del péptido señal que esté naturalmente asociada a la secuencia codificante del polipéptido que tiene actividad biológica
o puede ser una secuencia codificante del péptido señal diferente, tal como una de las descritas anteriormente.
[0068] En un aspecto preferido, la secuencia codificante del péptido señal es una secuencia naturalmente asociada a una secuencia codificante de beta-glucosidasa.
Enlaces
[0069] Tal y como se menciona supra, muchas endoglucanasas tienen una estructura multidominio que consiste en un dominio catalítico separado de uno o más módulos de unión a carbohidratos por un(os) péptido(s) enlazador(es). En los métodos de la presente invención, los constructos de proteína de fusión pueden además comprender una secuencia codificante de enlazador localizada 3' a la secuencia que comprende el dominio catalítico de endoglucanasa y 5' a la secuencia que comprende el dominio catalítico del polipéptido que tiene actividad biológica.
[0070] El enlazador se puede obtener a partir del mismo gen que el dominio catalítico de la endoglucanasa o a partir de un gen de endoglucanasa diferente. Por otro lado, el enlazador puede ser sintético en el origen.
[0071] Ejemplos de enlaces que se pueden usar en los métodos de la presente invención incluyen, pero de forma no limitativa, enlaces obtenidos a partir de los genes para la celobiohidrolasa I de Trichoderma reesei (Srisodsuk et al., 1993, Journal of Biological Chemistry 268: 20765-20761); celobiohidrolasa Cel7A de Hypocrea jecorina (precedentemente Trichoderma reesei) (Mulakala et al., 2005, Proteins 60: 598-605); endoglucanasa V de Humicola insolens; y endoglucanasa NRRL 8126 CEL7C Thielavia terrestris.
[0072] En un aspecto preferido, el enlazador se obtiene a partir de un gen de endoglucanasa de Humicola insolens. En otro aspecto preferido, el enlazador se obtiene a partir de un gen de endoglucanasa de Trichoderma reesei. En un aspecto más preferido, el enlazador se obtiene a partir de una endoglucanasa V del gen de Humicola insolens (eg5).
[0073] En otro aspecto preferido, el enlazador se obtiene a partir de un gen de endoglucanasa de Thielavia terrestris. En otro aspecto más preferido, el enlazador se obtiene a partir de un gen de endoglucanasa NRRL 8126 CEL7C de Thielavia terrestris.
[0074] En un aspecto preferido, el enlazador es al menos 5 residuos de aminoácidos. En un aspecto más preferido, el enlazador es al menos 15 residuos de aminoácidos. En un aspecto más preferido, el enlazador es al menos 25 residuos de aminoácidos.
[0075] En un aspecto preferido, el enlazador es entre aproximadamente 5 a aproximadamente 60 residuos de aminoácidos. En un aspecto más preferido, el enlazador es entre aproximadamente 15 a aproximadamente 50 residuos de aminoácidos. En un aspecto más preferido, el enlazador es entre aproximadamente 25 a aproximadamente 45 residuos de aminoácidos.
Módulos de unión a carbohidratos
[0076] Los módulos de unión a carbohidratos (CBMs) se definen como secuencias de aminoácidos contiguos con un pliegue discreto que tienen actividad de unión a carbohidratos y se encuentran comúnmente dentro de enzimas activas de carbohidratos. Varios tipos de CBMs se han descrito, y una mayoría de los mismos se enlazan a polisacáridos insolubles (véase Boraston et al., 2004, Biochem J. 382: 769-781). Se han caracterizado módulos de unión a carbohidratos que median interacción con, por ejemplo, celulosa cristalina, celulosa no cristalina, quitina, beta-1,3glucanos y glucanos de enlace beta-1,3-1,4-mezclado, xilano, manano, galactano y almidón. Los módulos de unión a carbohidratos ocurren frecuentemente en celulasas multidominio. Mientras algunos CBMs confieren enlace específico a un subconjunto de estructuras de carbohidratos, otros son más generales en su capacidad para asociarse con varios polisacáridos. Los CBMs que confieren unión a celulosa a veces se denominan dominios de enlace a celulosa, o CBDs (Boraston et al., 2004, Biochem J. 382: 769-781). Los CBMs se reagrupan por similitud de aminoácido; actualmente, hay descritas 48 familias de CBM.
[0077] Las glicósido hidrolasas pueden comprender más de un dominio catalítico y uno, dos, tres o más CBMs, y opcionalmente además comprenden una o varias regiones de secuencia de aminoácidos de polipéptido que conectan el
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ES07868319.0T 2006-07-21 2007-07-20 Métodos para aumentar la secreción de polipéptidos que tienen actividad biológica Active ES2538360T3 (es)

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US8735549B2 (en) 2014-05-27
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US8546106B2 (en) 2013-10-01
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