ES2330173A1 - Procedure of production and purification of the factor derived from the pigmented epithelium of the retina in a yeast system. (Machine-translation by Google Translate, not legally binding) - Google Patents
Procedure of production and purification of the factor derived from the pigmented epithelium of the retina in a yeast system. (Machine-translation by Google Translate, not legally binding) Download PDFInfo
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Abstract
Description
Procedimiento de producción y purificación del factor derivado del epitelio pigmentado de la retina en un sistema de levadura.Production procedure and purification of factor derived from the pigmented epithelium of the retina in a system of yeast
La presente invención está dentro del campo de la Biología Molecular y la Biotecnología. En particular, la invención se refiere a un procedimiento para producir y purificar la molécula completa de PEDF (Factor Derivado del Epitelio Pigmentado), y versiones mutadas y/o truncadas de dicha molécula en el sistema de expresión eucariota de Pichia pastoris. La presente invención también tiene como objeto la protección de las propias moléculas de PEDF directamente obtenidas mediante este procedimiento y el uso de las mismas en la preparación de composiciones farmacéuticas.The present invention is within the field of Molecular Biology and Biotechnology. In particular, the invention relates to a process for producing and purifying the entire PEDF molecule (Pigmented Epithelium Derived Factor), and mutated and / or truncated versions of said molecule in the eukaryotic expression system of Pichia pastoris . The present invention also has as its object the protection of the PEDF molecules themselves directly obtained by this procedure and the use thereof in the preparation of pharmaceutical compositions.
El factor PEDF fue inicialmente purificado a partir de medios condicionados de células epiteliales de la retina (Tombran-Tink, J. et al. 1991). PEDF es una proteína que inicialmente fue caracterizada como un inductor y regulador de la diferenciación celular, y se ha propuesto que PEDF podría regular la diferenciación neuronal en la retina embrionaria (Jablonski, M. et al., 1995). También se ha comprobado que PEDF ejerce actividad neurotrófica sobre todo tipo de células de origen neuronal, y además, aumenta la supervivencia de células granulosas del cerebelo en cultivo. También se ha descrito que esta proteína es uno de los factores anti-angiogénicos más potentes (Doll, J.A. et al., 2003) conocidos. Se han descrito en la molécula PEDF dos regiones:The PEDF factor was initially purified from conditioned media of epithelial cells of the retina (Tombran-Tink, J. et al . 1991). PEDF is a protein that was initially characterized as an inducer and regulator of cell differentiation, and it has been proposed that PEDF could regulate neuronal differentiation in the embryonic retina (Jablonski, M. et al ., 1995). It has also been shown that PEDF exerts neurotrophic activity on all types of cells of neuronal origin, and also increases the survival of cerebellar granule cells in culture. It has also been described that this protein is one of the most potent anti-angiogenic factors (Doll, JA et al ., 2003) known. Two regions have been described in the PEDF molecule:
- 1)one)
- Una región amino-terminal, actividad neurotrófica.A amino-terminal region, activity neurotrophic
- 2)2)
- Una región carboxilo-terminal, con actividad anti-angiogénica.A carboxyl-terminal region, with activity anti-angiogenic
El aislamiento de PEDF a partir de muestras (humor vítreo, por ejemplo, donde es abundante) es muy complicado debido a la dificultad de disponer de cantidad suficiente de tejido. Además, como se explicará a continuación, los procedimientos actuales de producción de esta proteína presentan importantes inconvenientes. Por ello, nuestro grupo de investigación consideró interesante expresar y purificar tanto la proteína completa como la región carboxilo de la misma, en la levadura Pichia pastoris, con el fin de analizar su posible efecto terapéutico en patologías tales como degeneración macular asociada a la edad, retinopatía diabética, cáncer, enfermedades neurodegenerativas, etc.The isolation of PEDF from samples (vitreous humor, for example, where it is abundant) is very complicated due to the difficulty of having enough tissue. In addition, as will be explained below, the current production procedures for this protein have important drawbacks. Therefore, our research group considered it interesting to express and purify both the complete protein and the carboxyl region thereof, in the yeast Pichia pastoris , in order to analyze its possible therapeutic effect in pathologies such as age-related macular degeneration, diabetic retinopathy, cancer, neurodegenerative diseases, etc.
La expresión y purificación del factor PEDF ha sido descrita con anterioridad en dos sistemas de producción diferentes:The expression and purification of the PEDF factor has previously described in two production systems different:
1) por una parte, se ha descrito su producción en la bacteria Escherichia coli (Becerra SP et al. 1993, JBC):1) on the one hand, its production has been described in Escherichia coli bacteria (Becerra SP et al . 1993, JBC):
- \bullet?
- USSN 07/952,796. A DNA Clones for the Expression of Pigment Epithelium Derived Growth Factor and Related Proteins. USSN 07 / 952,796. A DNA Clones for the Expression of Pigment Epithelium Derived Growth Factor and Related Proteins .
- \bullet?
- USSN 08/257,963. A Pigment Epithelium Derived Factor: Characterizations of Its Biological Activity and Sequences Encoding and Expressing the Protein. USSN 08/257,963. A Pigment Epithelium Derived Factor: Characterizations of Its Biological Activity and Sequences Encoding and Expressing the Protein .
- \bullet?
- USSN 08/279,979. A Retinal Pigmented Epithelium Derived Neurotrophic Factor. USSN 08 / 279,979. A Retinal Pigmented Epithelium Derived Neurotrophic Factor .
- \bullet?
- USSN 08/367,841. A Pigment Epithelium Derived Factor: Characterization, Genomic Organization and Sequence of the PEDF Gene. USSN 08 / 367,841. A Pigment Epithelium Derived Factor: Characterization, Genomic Organization and Sequence of the PEDF Gene .
- \bullet?
- USSN 08/377,710. A DNA Clones for the Expression of Pigment Epithelium Derived Factor and Related Proteins. USSN 08 / 377,710. A DNA Clones for the Expression of Pigment Epithelium Derived Factor and Related Proteins .
- \bullet?
- USSN 08/520,373. A Retinal Pigmented Epithelium Derived Neurotrophic Factor. USSN 08 / 520,373. A Retinal Pigmented Epithelium Derived Neurotrophic Factor .
Sin embargo, la expresión de PEDF en E.
coli publicada por este grupo de investigación presenta graves
problemas, tales como la no secreción de la proteína PEDF
producida, y su no solubilidad (presencia en cuerpos de inclusión),
lo que conlleva la incorporación de una serie de pasos en el
procedimiento de purificación (por ejemplo, la desnaturalización de
la proteína para su purificación, y su consiguiente paso de
renaturalización) que hacen a éste mucho más tedioso, además de
obtener la proteína en un estado conformacional que puede diferir
de su estado normal. Por otra parte, el hecho de producir esta
proteína en un organismo procariota, hace que modificaciones
post-traduccionales descritas en esta proteína, y
que son esenciales para su adecuada actividad (glicosilación,
fosforilación), no sean realizadas o lo sean de manera incorrecta,
obteniéndose por tanto una variante no "fisiológica" de la
proteína. Recientemente ha sido publicado por otro grupo de
investigación un procedimiento de producción de PEDF en E.
coli (Zhang T et al. 2005, Biotechnology Letters) que
solventa los problemas de falta de solubilidad y de secreción que
presentaba el procedimiento descrito por Becerra et al. 1993.
Este nuevo método incorpora además un paso final de digestión
enzimática con el fin de eliminar la molécula de Glutation
S-transferasa (GST), fusionada con el extremo
c-terminal de PEDF para facilitar la purificación
del factor PEDF. Este último paso complica aún más el procedimiento
de obtención de PEDF. Aún así, la proteína obtenida en un organismo
procariota no es seguro que sea madurada de forma adecuada, y esto
puede afectar de forma importante a la estructura y a la actividad
de la proteína obtenida.However, the expression of PEDF in E. coli published by this research group presents serious problems, such as the non-secretion of the PEDF protein produced, and its non-solubility (presence in inclusion bodies), which entails the incorporation of a series of steps in the purification procedure (for example, the denaturation of the protein for purification, and its consequent renaturation step) that make it much more tedious, in addition to obtaining the protein in a conformational state that may differ from its normal state On the other hand, the fact of producing this protein in a prokaryotic organism, causes that post-translational modifications described in this protein, and that are essential for its adequate activity (glycosylation, phosphorylation), are not carried out or are incorrectly, thus obtaining a non-"physiological" variant of the protein. Recently, a PEDF production procedure in E. coli (Zhang T et al . 2005, Biotechnology Letters) has been published by another research group that solves the problems of lack of solubility and secretion presented by the procedure described by Becerra et al. . 1993. This new method also incorporates a final step of enzymatic digestion in order to eliminate the Glutathione S-transferase (GST) molecule, fused with the end
c-terminal of PEDF to facilitate the purification of the PEDF factor. This last step further complicates the procedure for obtaining PEDF. Even so, the protein obtained in a prokaryotic organism is not sure that it is matured properly, and this can significantly affect the structure and activity of the protein obtained.
Por otra parte, en 1996 se describió la producción del factor PEDF en células eucariotas, concretamente, en cultivos de células de riñón de hámster (Stratikos E et al. 1996, Protein Science). La producción de PEDF en este tipo de células eucariotas, solventaba el importante problema de las modificaciones post-traduccionales, aunque incorporaba dos importantes inconvenientes al proceso de producción:On the other hand, in 1996 the production of the PEDF factor in eukaryotic cells was described, specifically, in hamster kidney cell cultures (Stratikos E et al . 1996, Protein Science). The production of PEDF in this type of eukaryotic cells, solved the important problem of post-translational modifications, although it incorporated two important drawbacks to the production process:
- a)to)
- El cultivo de líneas celulares es mucho más caro (medios de cultivo y suplementos necesarios, como suero fetal) que el sistema de producción en organismos unicelulares como E. coli.The cultivation of cell lines is much more expensive (culture media and necessary supplements, such as fetal serum) than the production system in unicellular organisms such as E. coli .
- b)b)
- El nivel de producción de PEDF en cultivos celulares es más bajo, encareciendo con ello todo el sistema de producción.He PEDF production level in cell cultures is lower, thereby increasing the entire production system.
La proteína comercial PEDF es producida en la actualidad principalmente en cultivos de células de riñón de hámster. Debido al elevado coste, este sistema de producción, y a los bajos niveles de proteína obtenida, la presente invención proporciona una nueva alternativa práctica, rápida, eficaz y mucho más económica para producir el factor PEDF. Por tanto, la presente invención propone un método o procedimiento de obtención del factor PEDF en un sistema de expresión diferente a los descritos en el estado de la técnica, fundamentado en la utilización de la levadura Pichia pastoris.The commercial PEDF protein is currently produced mainly in hamster kidney cell cultures. Due to the high cost, this production system, and the low levels of protein obtained, the present invention provides a new practical, fast, efficient and much cheaper alternative to produce the PEDF factor. Therefore, the present invention proposes a method or method of obtaining the PEDF factor in an expression system different from those described in the state of the art, based on the use of Pichia pastoris yeast.
La presente invención proporciona un nuevo procedimiento para producir el factor PEDF, un equivalente funcional del factor PEDF que contenga sustitución(es), adición(es), inserción(es) y/o deleción(es) únicas o múltiples en la secuencia de la proteína tipo salvaje y/o sustituciones de aminoácidos modificados químicamente que no afecten la función biológica, (así como versiones hiper- e hipo-fosforiladas que corresponden a las secuencias SEQ.ID.N.3, SEQ.ID.N.4, SEQ.ID.N.5 Y SEQ.ID.N.6 respectivamente), y/o versiones truncadas de PEDF en el sistema de expresión eucariota, preferentemente en levaduras y más concretamente en Pichia pastoris.The present invention provides a new method for producing the PEDF factor, a functional equivalent of the PEDF factor that contains single or multiple substitution (s), insertion (s) and / or deletion (s) in the sequence of the wild-type protein and / or chemically modified amino acid substitutions that do not affect biological function, (as well as hyper- and hypophosphorylated versions corresponding to the sequences SEQ.ID.N.3, SEQ.ID.N.4, SEQ .ID.N.5 AND SEQ.ID.N.6 respectively), and / or truncated versions of PEDF in the eukaryotic expression system, preferably in yeasts and more specifically in Pichia pastoris .
De acuerdo con la problemática planteada en líneas anteriores, estudios recientes (Maik-Rachline G. and Seger R., Blood., Dec. 2005) han descrito la modificación post-traduccional de PEDF (fosforilación), y cómo esta fosforilación afecta a las diferentes actividades de esta proteína. Como ya se indicado anteriormente, el empleo de células eucariotas para la expresar el factor PEDF favorece la obtención de las diferentes formas fosforiladas (por lo tanto funcionales) del mismo.According to the problem raised in previous lines, recent studies (Maik-Rachline G. and Seger R., Blood., Dec. 2005) have described the post-translational modification of PEDF (phosphorylation), and how this phosphorylation affects the Different activities of this protein. As already indicated previously, the use of eukaryotic cells to express the PEDF factor favors obtaining different forms phosphorylated (therefore functional) thereof.
Un aspecto muy importante de la presente invención es que la proteína producida en el sistema de Pichia pastoris es totalmente soluble, es secretada al medio de cultivo, y puede ser obtenida con un alto grado de pureza en un único paso cromatográfico, sin necesidad de incorporar ninguna etapa de digestión enzimática en el proceso de purificación.A very important aspect of the present invention is that the protein produced in the Pichia pastoris system is completely soluble, is secreted into the culture medium, and can be obtained with a high degree of purity in a single chromatographic step, without the need to incorporate No stage of enzymatic digestion in the purification process.
La ventaja fundamental que presenta el procedimiento descrito en la presente invención respecto al resto de procedimientos ya descritos es conseguir un sistema de producción sencillo y de bajo coste, que puede ser utilizado a nivel industrial para la producción de PEDF.The fundamental advantage of the procedure described in the present invention with respect to the rest of procedures already described is to achieve a system of simple and low cost production, which can be used at the level industrial for the production of PEDF.
Por lo tanto, según un primer aspecto importante, esta invención se refiere a un procedimiento para obtener factor PEDF aislado o purificado y/o un equivalente funcional del factor PEDF que contenga al menos una sustitución y/o una adición y/o una inserción y/o una deleción en la secuencia de la proteína tipo salvaje y/o contenga al menos una sustitución de aminoácidos modificados químicamente y/o versiones truncadas de PEDF en un sistema de expresión de una levadura que comprende las siguientes etapas:Therefore, according to a first aspect importantly, this invention relates to a method for obtain isolated or purified PEDF factor and / or an equivalent functional of the PEDF factor that contains at least one substitution and / or an addition and / or an insertion and / or a deletion in the sequence of the wild type protein and / or contain at least one substitution of chemically modified amino acids and / or truncated versions of PEDF in a yeast expression system comprising the following stages:
- a.to.
- Obtener el cADN de PEDF mediante RT-PCR partiendo de ARN total obtenido al menos a partir de células del epitelio pigmentado de retina;Obtain the PEDF cDNA by RT-PCR based on total RNA obtained at least at from retinal pigmented epithelium cells;
- b.b.
- Obtener mediante PCR versiones mutantes y/o truncadas, empleando como ADN molde la forma completa de PEDF;Obtain mutant versions using PCR and / or truncated, using as a template DNA the complete form of PEDF;
- c.C.
- Clonar las construcciones de la etapa a) y de la etapa b), en un vector de expresión de levaduras;Clone stage constructions a) and of step b), in an expression vector of yeasts;
- d.d.
- Transformar y seleccionar los clones de la levadura con las diferentes construcciones clonadas generadas en la etapa c);Transform and select clones of the yeast with the different cloned constructs generated in stage c);
- e.and.
- Inocular, con los clones obtenidos en la etapa d), el medio de crecimiento de levadura;Inoculate, with the clones obtained in step d), the yeast growth medium;
- f.F.
- Inducir el factor PEDF completo y/o truncado en la levadura en medio de cultivo de levaduras con metanol corno inductor;Induce the full PEDF factor and / or truncated in yeast in yeast culture medium with methanol inductor horn;
- g.g.
- Eliminar las células del cultivo mediante centrifugación;Remove the cells from the culture by centrifugation;
- h.h.
- Cambiar el medio de cultivo en el que están presentes el factor PEDF completo y/o truncado, a un tampón de unión apropiado para la cromatografía de afinidad;Change the culture medium in which the complete and / or truncated PEDF factor is present, to a buffer of appropriate binding for affinity chromatography;
- i.i.
- Purificar el factor PEDF completo y/o truncado, mediante cromatografía de afinidad;Purify the complete PEDF factor and / or truncated, by affinity chromatography;
- j.j.
- Cambiar el tampón del factor PEDF completo y/o truncado purificado a un tampón que permita su posterior empleo en estudios de actividad.Change the PEDF factor buffer complete and / or truncated purified to a buffer that allows its subsequent employment in activity studies.
La presente invención también se refiere al procedimiento para obtener factor PEDF aislado o purificado y/o un equivalente funcional del factor PEDF que contenga al menos una sustitución y/o una adición y/o una inserción y/o una deleción en la secuencia de la proteína tipo salvaje y/o contenga al menos una sustitución de aminoácidos modificados químicamente y/o versiones truncadas de PEDF según reivindicación 1 en un sistema de expresión de una levadura, preferentemente en Pichia pastoris que comprende las siguientes etapas:The present invention also relates to the process for obtaining isolated or purified PEDF factor and / or a functional equivalent of the PEDF factor containing at least one substitution and / or an addition and / or an insertion and / or a deletion in the sequence of the wild-type protein and / or contain at least one substitution of chemically modified amino acids and / or truncated versions of PEDF according to claim 1 in a yeast expression system, preferably in Pichia pastoris comprising the following steps:
- a.to.
- Obtener el cADN de PEDF mediante RT-PCR partiendo de ARN total obtenido al menos a partir de células del epitelio pigmentado de retina;Obtain the PEDF cDNA by RT-PCR based on total RNA obtained at least at from retinal pigmented epithelium cells;
- b.b.
- Obtener mediante PCR versiones mutantes y/o truncadas, empleando como ADN molde la forma completa de PEDF;Obtain mutant versions using PCR and / or truncated, using as a template DNA the complete form of PEDF;
- c.C.
- Clonar las construcciones de la etapa a) y de la etapa b), en un vector de expresión de Pichia pastoris;Clone the constructions of stage a) and stage b) into an expression vector of Pichia pastoris ;
- d.d.
- Transformar y seleccionar los clones de la levadura Pichia pastoris con las diferentes construcciones clonadas generadas en la etapa c);Transform and select the clones of the yeast Pichia pastoris with the different cloned constructs generated in stage c);
- e.and.
- Inocular, con los clones obtenidos, el medio de crecimiento de Pichia pastoris;Inoculate, with the clones obtained, the growth medium of Pichia pastoris ;
- f.F.
- Inducir el factor PEDF completo o truncado en la levadura en medio de cultivo de Pichia pastoris con metanol como inductor;Induce the complete or truncated PEDF factor in yeast in culture medium of Pichia pastoris with methanol as inducer;
- g.g.
- Eliminar las células del cultivo mediante centrifugación;Remove the cells from the culture by centrifugation;
- h.h.
- Cambiar el medio de cultivo en el que están presentes el factor PEDF completo y/o truncado, a un tampón de unión apropiado para la cromatografía de afinidad;Change the culture medium in which the complete and / or truncated PEDF factor is present, to a buffer of appropriate binding for affinity chromatography;
- i.i.
- Purificar el factor PEDF completo y/o truncado, cromatografía de afinidad;Purify the complete PEDF factor and / or truncated, affinity chromatography;
- j.j.
- Cambiar el tampón del factor PEDF completo y/o truncado purificado a un tampón que permita su posterior empleo en estudios de actividad.Change the PEDF factor buffer complete and / or truncated purified to a buffer that allows its subsequent employment in activity studies.
Según otro aspecto importante en la presente invención en la etapa c) el vector de expresión es pPICZ\alphaA.According to another important aspect here invention in step c) the expression vector is pPICZ?
Según otro aspecto importante en la presente invención en la etapa e) el medio de cultivo es el medio BMGY.According to another important aspect here invention in step e) the culture medium is the BMGY medium.
Según otro aspecto importante en la presente invención en la etapa j) el tampón que permite el posterior empleo en estudio de actividad es tampón PBS.According to another important aspect here invention in step j) the buffer that allows subsequent use in activity study it is PBS buffer.
Según otro aspecto importante en la presente invención en la etapa a) los cebadores empleados se seleccionan de la secuencias SEQ.ID.N.7 y/o SEQ.ID.N.8 y/o cualquier cebador equivalente cuya secuencia nucleotídica solape con las secuencias SEQ.ID.N.7 y/o SEQ.ID.N.8.According to another important aspect here invention in step a) the primers used are selected from the sequences SEQ.ID.N.7 and / or SEQ.ID.N.8 and / or any primer equivalent whose nucleotide sequence overlaps the sequences SEQ.ID.N.7 and / or SEQ.ID.N.8.
Según otro aspecto importante en la presente invención en la etapa b) los cebadores empleados se seleccionan del grupo formado por la secuencias SEQ.ID.N.9, SEQ.ID.N.10, SEQ.ID.N.11, SEQ.ID.N.12, SEQ.ID.N.13, SEQ.ID.N.14, SEQ.ID.N.15, SEQ.ID.N.16, SEQ.ID.N.17, SEQ.ID.N.18, SEQ.ID.N.19, SEQ.ID.N.20, SEQ.ID.N.21, SEQ.ID.N.22 y/o cualquier cebador equivalente cuya secuencia nucleotídica solape con las secuencias SEQ.ID.N.9, SEQ.ID.N.10, SEQ.ID.N.1 1, SEQ.ID.N.12, SEQ.ID.N.13, SEQ.ID.N.14, SEQ.ID.N.15, SEQ.ID.N.16, SEQ.ID.N.17, SEQ.ID.N.18, SEQ.ID.N.19, SEQ.ID.N.20, SEQ.ID.N.21, SEQ.ID.N.22.According to another important aspect here invention in step b) the primers used are selected from the group consisting of the sequences SEQ.ID.N.9, SEQ.ID.N.10, SEQ.ID.N.11, SEQ.ID.N.12, SEQ.ID.N.13, SEQ.ID.N.14, SEQ.ID.N.15, SEQ.ID.N.16, SEQ.ID.N.17, SEQ.ID.N.18, SEQ.ID.N.19, SEQ.ID.N.20, SEQ.ID.N.21, SEQ.ID.N.22 and / or any equivalent primer whose nucleotide sequence overlaps with sequences SEQ.ID.N.9, SEQ.ID.N.10, SEQ.ID.N.1 1, SEQ.ID.N.12, SEQ.ID.N.13, SEQ.ID.N.14, SEQ.ID.N.15, SEQ.ID.N.16, SEQ.ID.N.17, SEQ.ID.N.18, SEQ.ID.N.19, SEQ.ID.N.20, SEQ.ID.N.21, SEQ.ID.N.22.
Según otro aspecto importante en la presente invención en la etapa b) los sitios de restricción utilizados son EcoRI y XbaI.According to another important aspect here invention in step b) the restriction sites used are EcoRI and XbaI.
Según otro aspecto importante en la presente invención se han empleado unas cepas GS-115 y X-33.According to another important aspect here invention GS-115 strains have been used and X-33
Según otro aspecto importante en la presente invención en la etapa b) la inducción de PEDF en Pichia pastoris se realiza a un intervalo de temperatura entre 25-30ºC.According to another important aspect in the present invention in step b) the induction of PEDF in Pichia pastoris is carried out at a temperature range between 25-30 ° C.
Según otro aspecto importante en la presente invención en la etapa e) se ha empleado un 2% de PMSF.According to another important aspect here invention in step e) 2% of PMSF has been used.
Según un segundo aspecto importante la presente invención se refiere al producto obtenido directamente a partir del procedimiento descrito en la presente invención en particular al factor PEDF aislado o purificado que comprende una secuencia de aminoácidos la cual tiene al menos un 80% de identidad con la secuencia SEQ.ID.N.1. sobre toda su longitud, al factor PEDF aislado o purificado que comprende una secuencia de aminoácidos la cual tiene al menos un 80% de identidad con la secuencia SEQ.ID.N.2. sobre toda su longitud, al factor PEDF aislado o purificado que comprende una secuencia de aminoácidos la cual tiene al menos un 80% de identidad con la secuencia SEQ.ID.N.3. sobre toda su longitud, al factor PEDF aislado o purificado que comprende una secuencia de aminoácidos la cual tiene al menos un 80% de identidad con la secuencia SEQ.ID.N.4. sobre toda su longitud, al factor PEDF aislado o purificado que comprende una secuencia de aminoácidos la cual tiene al menos un 80% de identidad con la secuencia SEQ.ID.N.5. sobre toda su longitud, al factor PEDF aislado o purificado que comprende una secuencia de aminoácidos la cual tiene al menos un 80% de identidad con la secuencia SEQ.ID.N.6. sobre toda su longitud,. Y de forma más específica al factor PEDF aislado o purificado que consiste en una secuencia aminoacídica escogida del grupo formado por las secuencias SEQ.ID.N.1 a SEQ.ID.N.6, por sus secuencias complementarias y/o por sus secuencias equivalentes funcionales.According to a second important aspect, the present invention refers to the product obtained directly from the procedure described in the present invention in particular at isolated or purified PEDF factor comprising a sequence of amino acids which has at least 80% identity with the sequence SEQ.ID.N.1. over its entire length, to the PEDF factor isolated or purified comprising an amino acid sequence the which has at least 80% identity with the sequence SEQ.ID.N.2. over its entire length, to the isolated PEDF factor or purified comprising an amino acid sequence which has at least 80% identity with the sequence SEQ.ID.N.3. on full length, to the isolated or purified PEDF factor comprising an amino acid sequence which has at least 80% of identity with the sequence SEQ.ID.N.4. over its entire length, at isolated or purified PEDF factor comprising a sequence of amino acids which has at least 80% identity with the sequence SEQ.ID.N.5. over its entire length, to the PEDF factor isolated or purified comprising an amino acid sequence the which has at least 80% identity with the sequence SEQ.ID.N.6. over its entire length ,. And more specifically to the PEDF factor isolated or purified consisting of an amino acid sequence chosen from the group formed by the sequences SEQ.ID.N.1 a SEQ.ID.N.6, for its complementary sequences and / or for its functional equivalent sequences.
Según tercer aspecto de la presente invención se refiere al uso del factor PEDF en la preparación de un medicamento, y al uso del factor PEDF de secuencias SEQ.ID.N.2, SEQ.ID.N.5 Y SEQ.ID.N.6 en la preparación de una composición que tiene efecto antagonista de PEDF de SEQ.ID.N.1, SEQ.ID.N.3 Y SEQ.ID.N.4 y al uso de una composición que inhibe la diferenciación celular, al uso del factor PEDF en la preparación de una composición química que inhibe el efecto de las secuencias SEQ.ID.N.1, SEQ.ID.N.2 Y SEQ.ID.N.3., al uso del factor PEDF en la preparación de una composición que inhibe la diferenciación celular y al uso del factor PEDF en la preparación de una composición química que estimula la diferenciación celular que contiene las secuencias SEQ.ID.N.1, SEQ.ID.N.3 Y SEQ.ID.N.4.According to the third aspect of the present invention, refers to the use of the PEDF factor in the preparation of a medicine, and the use of the sequence PEDF factor SEQ.ID.N.2, SEQ.ID.N.5 Y SEQ.ID.N.6 in the preparation of a composition that has an effect PEDF antagonist of SEQ.ID.N.1, SEQ.ID.N.3 AND SEQ.ID.N.4 and to use of a composition that inhibits cell differentiation, when using PEDF factor in the preparation of a chemical composition that inhibits the effect of the sequences SEQ.ID.N.1, SEQ.ID.N.2 AND SEQ.ID.N.3., to the use of the PEDF factor in the preparation of a composition that inhibits cell differentiation and the use of the PEDF factor in the preparation of a chemical composition that stimulates the cell differentiation containing the sequences SEQ.ID.N.1, SEQ.ID.N.3 AND SEQ.ID.N.4.
La molécula completa de PEDF (SEQ.ID.N.1) obtenida según el procedimiento anteriormente descrito incrementa la diferenciación neuronal en células madre.The complete PEDF molecule (SEQ.ID.N.1) obtained according to the procedure described above increases Neural differentiation in stem cells.
Se obtiene de forma adicional una molécula truncada de PEDF (SEQ.ID.N.2), que, obtenida según el procedimiento anteriormente descrito, es capaz de inhibir los efectos producidos por la molécula completa de PEDF. Por lo tanto el presente procedimiento proporciona dos productos relacionados, la molécula truncada de PEDF y el PEDF completo, guardando por tanto la presente invención un mismo concepto inventivo.An additional molecule is obtained truncated of PEDF (SEQ.ID.N.2), which, obtained according to the procedure previously described, it is able to inhibit the effects produced for the complete PEDF molecule. Therefore the present procedure provides two related products, the molecule truncated of PEDF and the complete PEDF, thus saving the Present invention the same inventive concept.
Los siguientes ejemplos específicos que se proporcionan sirven para ilustrar la naturaleza de la presente invención. Estos ejemplos se incluyen solamente con fines ilustrativos y no han de ser interpretados como limitaciones a la invención que aquí se reivindica.The following specific examples to be provide serve to illustrate the nature of the present invention. These examples are included for purposes only. illustrative and should not be construed as limitations to the invention claimed here.
Se ha clonado el cDNA (región codificante) de PEDF (obtenido mediante RT-PCR) en el vector de expresión pPICZ\alphaA utilizando los sitios de restricción EcoRI y XbaI. La ausencia de mutaciones ha sido confirmada mediante secuenciación automática de ADN. También se ha clonado y producido una forma truncada, correspondiente al extremo carboxilo terminal (aminoácidos 195-400). Estas moléculas han sido diseñadas de forma que incorporan en su extremo C-terminal los epítopos c-myc e His6x, para facilitar su detección y purificación.The cDNA (coding region) of PEDF (obtained by RT-PCR) in the vector of pPICZαA expression using EcoRI restriction sites and XbaI. The absence of mutations has been confirmed by automatic DNA sequencing It has also been cloned and produced a truncated form, corresponding to the carboxyl terminal end (amino acids 195-400). These molecules have been designed so that they incorporate at its end C-terminal epitopes c-myc e His6x, to facilitate its detection and purification.
Las construcciones generadas fueron utilizadas para transformar diferentes cepas de P. pastoris, empleando para ello el kit "Easy select Pichia expresión kit" (Invitrogen).The generated constructs were used to transform different strains of P. pastoris , using the "Easy select Pichia expression kit" (Invitrogen).
Se analizó la producción en dos cepas distintas, GS-115 y X-33, después de crecer un inóculo inicial de cada clon en BMGY durante 48 horas, y de inducir posteriormente la expresión durante 48 h en medio BMMY, utilizando metanol como agente inductor. Concluida la inducción, las células fueron separadas del medio de cultivo mediante centrifugación. La confirmación de la expresión de estas proteínas fue analizada mediante western blot de muestras del medio de cultivo, utilizando un anticuerpo anti-myc. En total se analizaron 5 clones de la cepa GS-115 y 4 de la cepa X-33. Se observaron diferencias importantes en el nivel de producción de la proteína recombinante entre los diferentes clones, como se muestra en la figura 1. Se seleccionó para la producción el clon 2 de la cepa X-33 por ser el que ofreció un mayor nivel de producción. En la figura 1 se puede observar la detección mediante western blot de PEDF en el medio de cultivo de distintos clones recombinantes de Pichia pastoris.Production was analyzed in two different strains, GS-115 and X-33, after growing an initial inoculum of each clone in BMGY for 48 hours, and subsequently inducing expression for 48 h in BMMY medium, using methanol as inducing agent . Upon completion of the induction, the cells were separated from the culture medium by centrifugation. Confirmation of the expression of these proteins was analyzed by western blot of samples from the culture medium, using an anti-myc antibody. In total, 5 clones of strain GS-115 and 4 of strain X-33 were analyzed. Important differences were observed in the production level of the recombinant protein between the different clones, as shown in Figure 1. Clone 2 of strain X-33 was selected for production because it offered the highest production level. . Figure 1 shows the detection by western blot of PEDF in the culture medium of different recombinant clones of Pichia pastoris .
En la figura 2 se muestra la expresión en Pichia pastoris de los clones seleccionados para la expresión de la forma completa y truncada PEDF, y detección mediante western blot.Figure 2 shows the expression in Pichia pastoris of the clones selected for the expression of the form complete and truncated PEDF, and detection by western blot.
Las proteínas recombinantes obtenidas por el procedimiento anterior, han sido purificadas mediante cromatografía de afinidad. La figura 3 muestra el análisis electroforético de las distintas fracciones obtenidas en la cromatografía de la molécula de PEDF completa. La proteína recombinante, tanto la forma completa como la truncada, una vez purificadas, fueron cuantificadas mediante el método Bradford, obteniéndose una concentración de aproximadamente 30 \mugr de PEDF/ml de medio de cultivo, y 20 \mugr de C-terminal/ml de medio de cultivo. Mediante electroforesis se estimó que la pureza de la preparación obtenida era cercana al 100%. La figura 3 muestra exactamente el análisis mediante electroforesis en gel de poliacrilamida (10%) en presencia de SDS, de las fracciones obtenidas en la purificación de PEDF completa recombinante mediante cromatografía de afinidad. El gel fue teñido con Comassie Blue. 1: fracción no retenida; 2 y 3: fracciones de lavado de la columna; 4 y 5: fracciones de elución. Observe la ausencia de bandas distintas a PEDF, lo que indica la elevada pureza de la proteína PEDF obtenida en la fracción 5.The recombinant proteins obtained by the previous procedure, have been purified by chromatography of affinity Figure 3 shows the electrophoretic analysis of the different fractions obtained in the chromatography of the molecule of complete PEDF. The recombinant protein, both the complete form as the truncated, once purified, were quantified using the Bradford method, obtaining a concentration of approximately 30 µg PEDF / ml culture medium, and 20 µg of C-terminal / ml of culture medium. The purity of the preparation was estimated by electrophoresis obtained was close to 100%. Figure 3 shows exactly the polyacrylamide gel electrophoresis analysis (10%) in presence of SDS, of the fractions obtained in the purification of Complete PEDF recombinant by affinity chromatography. He gel was stained with Comassie Blue. 1: fraction not retained; 2 and 3: column wash fractions; 4 and 5: elution fractions. Note the absence of bands other than PEDF, which indicates the high purity of the PEDF protein obtained in fraction 5.
Las proteínas purificadas según el método descrito (tanto la forma completa como la versión truncada) son totalmente solubles. Se evita por tanto el empleo de urea, o de otros agentes solubilizantes, que podrían interferir en la actividad de la proteína, o impedir su uso directo en ensayos, y que obligaría a añadir pasos adicionales para la eliminación de estos agentes.The purified proteins according to the method described (both the complete form and the truncated version) are totally soluble. The use of urea, or of other solubilizing agents, which could interfere with activity of the protein, or prevent its direct use in assays, and that would force you to add additional steps to remove these Agents
Posteriormente se procedió al cambio del tampón de elución a PBS, empleando el sistema de ultrafiltración/diálisis, en el que la proteína purificada es totalmente soluble. Una vez en este tampón, se realizaron los ensayos para analizar la actividad de la proteína obtenida.Subsequently, the buffer was changed elution to PBS, using the ultrafiltration / dialysis system, in which the purified protein is totally soluble. Once in This buffer assays were performed to analyze the activity of The protein obtained.
Se han llevado a cabo estudios para comprobar que la proteína PEDF obtenida según el procedimiento anteriormente descrito presenta actividad potenciadora de la diferenciación neuronal, además de su papel en supervivencia como factor neurotrófico, previamente descrito en la literatura, así como la actividad de la molécula truncada (C-terminal) de PEDF, como molécula inhibidora de la diferenciación neuronal. Para ello se cultivaron células madre neurales embrionarias derivadas de la pared del ventrículo lateral procedentes de embriones de ratón de estadio E14.5, en presencia y ausencia del factor PEDF, o de C-terminal, obtenidos según el procedimiento descrito anteriormente. El protocolo de diferenciación de estas células se llevó a cabo basándose en los protocolos de diferenciación previamente descritos para este tipo celular que constan de un periodo de tiempo en cultivo con medio DMEM-F12 enriquecido con N2 suplemento, al que se añade 10 ng/ml de bFGF durante los dos primeros días del cultivo, y 2% de FBS durante los cinco días siguientes del cultivo. Transcurridos los dos primeros días, el medio en el que se cultivan las células se cambio en todos los casos a DMEM-F12 conteniendo N2 supplement y con 2% de FBS durante los cinco días siguientes. Los resultados obtenidos, recogidos en la figura 4 (recuento de células tuj-1 positivas, es decir, neuronas, presentes en el cultivo) muestran que la proteína PEDF obtenida con el protocolo aquí expuesto es capaz de incrementar el número de neuronas presentes en el cultivo, indicando que la molécula obtenida es funcional para esta actividad (***: p<0.001). Por otra parte, estos resultados muestran que la molécula C-terminal de PEDF es capaz de inhibir el número de neuronas presentes en el cultivo, indicando que esta molécula presenta esta actividad (*: p<0.05). En la figura 4 se muestra el recuento de células neuronales o número de neuronas diferenciadas, en la que A viene definido por el ensayo control, B está definido por en ensayo con PEDF y C está definido por el ensayo con la versión truncada C-terminal, en el que se precia cómo el ensayo B aumenta el porcentaje de neuronas diferenciadas y cómo el efecto del ensayo en el se adiciona versión truncada C-terminal (C) disminuye el número de neuronas diferenciadas y por tanto presenta un efecto antagónico o inhibitorio de los efectos producidos por PEDF. También en la figura 4, D representa la diferenciación neuronal en la muestra control y en E se observa claramente cómo aumenta la diferenciación neuronal, cuando se adiciona PEDF.Studies have been carried out to verify that the PEDF protein obtained according to the procedure described above has neuronal differentiation enhancing activity, in addition to its survival role as a neurotrophic factor, previously described in the literature, as well as the activity of the truncated molecule (C-terminal) of PEDF, as a molecule that inhibits neuronal differentiation . For this, embryonic neural stem cells derived from the lateral ventricle wall from E14.5 stage mouse embryos were cultured, in the presence and absence of the PEDF factor, or C-terminal, obtained according to the procedure described above. The differentiation protocol of these cells was carried out based on the differentiation protocols previously described for this cell type that consist of a period of time in culture with DMEM-F12 medium enriched with N2 supplement, to which 10 ng / ml is added of bFGF during the first two days of the culture, and 2% of FBS during the five following days of the culture. After the first two days, the medium in which the cells are grown was changed in all cases to DMEM-F12 containing N2 supplement and with 2% FBS for the next five days. The results obtained, collected in Figure 4 (count of positive tuj-1 cells, that is, neurons, present in the culture) show that the PEDF protein obtained with the protocol set forth herein is capable of increasing the number of neurons present in the culture, indicating that the molecule obtained is functional for this activity (***: p <0.001). On the other hand, these results show that the C-terminal molecule of PEDF is able to inhibit the number of neurons present in the culture, indicating that this molecule has this activity (*: p <0.05). Figure 4 shows the neuronal cell count or number of differentiated neurons, in which A is defined by the control assay, B is defined by an assay with PEDF and C is defined by the assay with the truncated C-terminal version , in which it is appreciated how test B increases the percentage of differentiated neurons and how the effect of the test on the truncated C-terminal version (C) is added decreases the number of differentiated neurons and therefore presents an antagonistic or inhibitory effect of the effects produced by PEDF. Also in Figure 4, D represents the neuronal differentiation in the control sample and in E it is clearly seen how neuronal differentiation increases, when PEDF is added.
Mediante este sistema de producción se obtiene de forma eficiente y rápida tanto la forma completa de PEDF recombinante, como de su región C-terminal (aminoácidos 191-400) en la levadura Pichia pastoris, y gracias a la metodología posteriormente empleada para la purificación de las proteínas recombinantes obtenidas (cromatografía de afinidad) se obtienen aproximadamente 30 \mugr de proteína PEDF completa por ml de medio de cultivo, y 20 \mugr de proteína C-terminal, constituyendo una solución práctica a la problemática actual relativa a la obtención de poca cantidad de proteína soluble en un sistema eucariota de producción.Through this production system, the complete form of recombinant PEDF, as well as its C-terminal region (amino acids 191-400) in the yeast Pichia pastoris , is obtained efficiently and quickly, and thanks to the methodology subsequently used for the purification of The recombinant proteins obtained (affinity chromatography) are obtained approximately 30 µg of complete PEDF protein per ml of culture medium, and 20 µgr of C-terminal protein, constituting a practical solution to the current problem related to obtaining little amount of soluble protein in a eukaryotic production system.
<110> Universidad de Castilla-La Mancha<110> University of Castilla la Mancha
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<120> PROCEDIMIENTO DE PRODUCCIÓN Y PURIFICACIÓN DEL FACTOR DERIVADO DEL EPITELIO PIGMENTADO DE LA RETINA (PEDF) EN UN SISTEMA DE LEVADURA<120> PRODUCTION PROCEDURE AND PURIFICATION OF THE FACTOR DERIVED FROM THE PIGMENTED EPITEL OF THE RETINA (PEDF) IN A LIFTING SYSTEM
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<130> P340/2006<130> P340 / 2006
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<140> 2006-0101<140> 2006-0101
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<141> 2006-02-17<141> 2006-02-17
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<170> PatentIn version 3.3<170> PatentIn version 3.3
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<213> Artificial<213> Artificial
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<220><220>
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<223> PEDF<223> PEDF
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<220><220>
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<223> PEDF C-TERMINAL<223> C-TERMINAL PEDF
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<212> PRT<212> PRT
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<220><220>
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<223> PEDF-HIPERFOSFORILADO<223> PEDF-HYPERPHOSPHORILED
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<212> PRT<212> PRT
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<223> PEDF-HIPOFOSFORILADO<223> PEDF-HYPOPHOSPHORILED
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<223> PEDF-C-TER-HIPERFOSFORILADO<223> PEDF-C-TER-HYPERPHOSPHORILED
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<212> PRT<212> PRT
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<223> PEDF-C-TER-HIPOFOSFORILADO<223> PEDF-C-TER-HIPOFOSFORILADO
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<212> DNA<212> DNA
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<223> CEBADOR-1<223> PRIMER-1
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<223> CEBADOR-2<223> PRIMER-2
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<210> 9<210> 9
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 30<211> 30
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Artificial<213> Artificial
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> CEBADOR-3<223> CEBADOR-3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 9<400> 9
\hskip1cm\ hskip1cm
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 10<210> 10
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 30<211> 30
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Artificial<213> Artificial
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> CEBADOR-4<223> CEBADOR-4
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 10<400> 10
\hskip1cm\ hskip1cm
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 11<210> 11
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 29<211> 29
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Artificial<213> Artificial
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> ser24ala UP<223> ser24ala UP
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 11<400> 11
\hskip1cm\ hskip1cm
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 12<210> 12
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 29<211> 29
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Artificial<213> Artificial
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> ser24ala DW<223> ser24ala DW
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 12<400> 12
\hskip1cm\ hskip1cm
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 13<210> 13
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 31<211> 31
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Artificial<213> Artificial
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> ser24glu UP<223> ser24glu UP
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 13<400> 13
\hskip1cm\ hskip1cm
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 14<210> 14
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 31<211> 31
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Artificial<213> Artificial
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> ser24glu DW<223> ser24glu DW
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 14<400> 14
\hskip1cm\ hskip1cm
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 15<210> 15
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 38<211> 38
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Artificial<213> Artificial
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> ser114ala UP<223> ser114ala UP
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 15<400> 15
\hskip1cm\ hskip1cm
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 16<210> 16
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 38<211> 38
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Artificial<213> Artificial
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> ser114ala DW<223> ser114ala DW
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 16<400> 16
\hskip1cm\ hskip1cm
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 17<210> 17
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 41<211> 41
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Artificial<213> Artificial
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> ser114glu UP<223> ser114glu UP
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 17<400> 17
\hskip1cm\ hskip1cm
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 18<210> 18
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 41<211> 41
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Artificial<213> Artificial
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> ser114glu DW<223> ser114glu DW
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 18<400> 18
\hskip1cm\ hskip1cm
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 19<210> 19
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 37<211> 37
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Artificial<213> Artificial
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> ser227ala UP<223> ser227ala UP
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 19<400> 19
\hskip1cm\ hskip1cm
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 20<210> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 37<211> 37
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Artificial<213> Artificial
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> ser227ala DW<223> ser227ala DW
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 20<400> 20
\hskip1cm\ hskip1cm
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 21<210> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 41<211> 41
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Artificial<213> Artificial
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> ser227glu UP<223> ser227glu UP
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 21<400> 21
\hskip1cm\ hskip1cm
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 22<210> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 41<211> 41
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Artificial<213> Artificial
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> ser227glu DW<223> ser227glu DW
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 22<400> 22
\hskip1cm\ hskip1cm
Claims (20)
- a)to)
- Obtener el cADN de PEDF mediante RT-PCR partiendo de ARN total obtenido al menos a partir de células del epitelio pigmentado de retina;Obtain the PEDF cDNA by RT-PCR based on total RNA obtained at least at from retinal pigmented epithelium cells;
- b)b)
- Obtener mediante PCR versiones mutantes y/o truncadas, empleando como ADN molde la forma completa de PEDF;Obtain mutant versions using PCR and / or truncated, using as a template DNA the complete form of PEDF;
- c)C)
- Clonar las construcciones de la etapa a) y de la etapa b), en un vector de expresión de levaduras;Clone stage constructions a) and of step b), in an expression vector of yeasts;
- d)d)
- Transformar y seleccionar los clones de la levadura con las diferentes construcciones clonadas generadas en la etapa c);Transform and select clones of the yeast with the different cloned constructs generated in stage c);
- e)and)
- Inocular, con los clones obtenidos en la etapa d), el medio de crecimiento de levadura;Inoculate, with the clones obtained in step d), the yeast growth medium;
- f)F)
- Inducir el factor PEDF completo y/o truncado en la levadura en medio de cultivo de levaduras con metanol como inductor;Induce the full PEDF factor and / or truncated in yeast in yeast culture medium with methanol as inducer;
- g)g)
- Eliminar las células del cultivo mediante centrifugación;Remove the cells from the culture by centrifugation;
- h)h)
- Cambiar el medio de cultivo en el que están presentes el factor PEDF completo y/o truncado, a un tampón de unión apropiado para la cromatografía de afinidad;Change the culture medium in which the complete and / or truncated PEDF factor is present, to a buffer of appropriate binding for affinity chromatography;
- i)i)
- Purificar el factor PEDF completo y/o truncado, mediante cromatografía de afinidad;Purify the complete PEDF factor and / or truncated, by affinity chromatography;
- j)j)
- Cambiar el tampón del factor PEDF completo y/o truncado purificado a un tampón que permita su posterior empleo en estudios de actividad.Change the PEDF factor buffer complete and / or truncated purified to a buffer that allows its subsequent employment in activity studies.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\newpage\ newpage
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200600385A ES2330173B1 (en) | 2006-02-17 | 2006-02-17 | PROCEDURE OF PRODUCTION AND PURIFICATION OF THE FACTOR DERIVED FROM THE RETINA PIGMENTED EPITHEL IN A LIFTING SYSTEM. |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200600385A ES2330173B1 (en) | 2006-02-17 | 2006-02-17 | PROCEDURE OF PRODUCTION AND PURIFICATION OF THE FACTOR DERIVED FROM THE RETINA PIGMENTED EPITHEL IN A LIFTING SYSTEM. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| ES2330173A1 true ES2330173A1 (en) | 2009-12-04 |
| ES2330173B1 ES2330173B1 (en) | 2010-10-13 |
Family
ID=41328835
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES200600385A Active ES2330173B1 (en) | 2006-02-17 | 2006-02-17 | PROCEDURE OF PRODUCTION AND PURIFICATION OF THE FACTOR DERIVED FROM THE RETINA PIGMENTED EPITHEL IN A LIFTING SYSTEM. |
Country Status (1)
| Country | Link |
|---|---|
| ES (1) | ES2330173B1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006054278A2 (en) * | 2004-11-16 | 2006-05-26 | Yeda Research And Development Co. Ltd. At The Weizmann Institute Of Science | Variants of pigment epithelium derived factor and uses thereof |
-
2006
- 2006-02-17 ES ES200600385A patent/ES2330173B1/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006054278A2 (en) * | 2004-11-16 | 2006-05-26 | Yeda Research And Development Co. Ltd. At The Weizmann Institute Of Science | Variants of pigment epithelium derived factor and uses thereof |
Non-Patent Citations (4)
| Title |
|---|
| INVITROGEN, Life Technologies. "{}Easy Select TM Pichia Expression Kit"{}. A Manual of Methods for Expression of Recombinant Proteins Using pPICZ and pPICZ? in Pichia pastoris. Catalog no. K1740-01; 01.11.2005; [en línea]; [recuperado el 20.10.2008]. Recuperado de Internet <URL: http://products.invitrogen.com/ivgn/en/US/ adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect& productID=K174001&CID=Search-K1740-01&messageType=catProduc Detail&showAddButton=true >. Todo el documento, especialment páginas 14-19. * |
| INVITROGEN, Life Technologies. "Easy Select TM Pichia Expression Kit". A Manual of Methods for Expression of Recombinant Proteins Using pPICZ and pPICZ? in Pichia pastoris. Catalog no. K1740-01; 01.11.2005; [en línea]; [recuperado el 20.10.2008]. Recuperado de Internet <URL: http://products.invitrogen.com/ivgn/en/US/ adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect& productID=K174001&CID=Search-K1740-01&messageType=catProduct Detail&showAddButton=true >. Todo el documento, especialmente páginas 14-19. * |
| STEELE, F.R. "{}Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family"{}. PROC. NATL. ACAD. SCI. USA. Febrero 1992. Vol. 90, páginas 1526-1530; todo el documento. * |
| STEELE, F.R. "Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family". PROC. NATL. ACAD. SCI. USA. Febrero 1992. Vol. 90, páginas 1526-1530; todo el documento. * |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2330173B1 (en) | 2010-10-13 |
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