ES2342707B1 - BONE MORPHOGENETIC PROTEIN-2 (BMP-2) WITH A SPECIFIC UNION DOMAIN TO TYPE I COLLAGEN, PROCEDURE OF OBTAINING AND APPLICATIONS. - Google Patents
BONE MORPHOGENETIC PROTEIN-2 (BMP-2) WITH A SPECIFIC UNION DOMAIN TO TYPE I COLLAGEN, PROCEDURE OF OBTAINING AND APPLICATIONS. Download PDFInfo
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- ES2342707B1 ES2342707B1 ES200900050A ES200900050A ES2342707B1 ES 2342707 B1 ES2342707 B1 ES 2342707B1 ES 200900050 A ES200900050 A ES 200900050A ES 200900050 A ES200900050 A ES 200900050A ES 2342707 B1 ES2342707 B1 ES 2342707B1
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- rhbmp
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Peptides Or Proteins (AREA)
Abstract
Proteína morfogenética de hueso-2 (BMP-2) con un dominio de unión específico a colágeno tipo I, procedimiento de obtención y aplicaciones.Morphogenetic protein of bone-2 (BMP-2) with a domain of specific binding to type I collagen, procedure for obtaining and Applications.
La presente invención comprende la producción y aplicación de una proteína de fusión que consiste en una rhBMP-2 modificada para unirse específicamente a colágeno con un dominio de unión a colágeno derivado del factor de von Willebrand. La proteína de fusión resultante (rhBMP-2-CBD) presenta mayor afinidad por el colágeno, siendo esa unión muy estable en el tiempo, y reteniendo la actividad osteoinductiva, siendo capaz se inducir la formación de hueso nuevo incluso a concentraciones por debajo de las conocidas para la rhBMP-2 nativa. Además, en combinación con un transportador colagénico libre de ningún otro factor de crecimiento, como una esponja absorbible de colágeno, bajas concentraciones de rhBMP-2-CBD son capaces de inducir la formación de hueso nuevo cuando es implantada in vivo.The present invention comprises the production and application of a fusion protein consisting of a rhBMP-2 modified to specifically bind collagen with a collagen binding domain derived from von Willebrand factor. The resulting fusion protein (rhBMP-2-CBD) has a higher affinity for collagen, this binding being very stable over time, and retaining osteoinductive activity, being able to induce the formation of new bone even at concentrations below known for the native rhBMP-2. In addition, in combination with a collagen transporter free of any other growth factor, such as a collagen absorbable sponge, low concentrations of rhBMP-2-CBD are capable of inducing new bone formation when implanted in vivo .
Description
Proteína morfogenética de hueso-2 (BMP-2) con un dominio de unión específico a colágeno tipo I, procedimiento de obtención y aplicaciones.Morphogenetic protein of bone-2 (BMP-2) with a domain of specific binding to type I collagen, procedure for obtaining and Applications.
La presente invención se enmarca dentro de áreas de conocimiento tales como la biología, la biotecnología, la ciencia de materiales, la farmacología, la medicina, la ingeniería o la nanotecnología entre otras. Sus aplicaciones van destinadas fundamentalmente a los sectores de la salud y la biomedicina, pudiéndose emplear en la mejora de tratamientos y terapias en medicina regenerativa de tejidos óseos.The present invention is framed within areas of knowledge such as biology, biotechnology, science of materials, pharmacology, medicine, engineering or nanotechnology among others. Your applications are intended fundamentally to the health and biomedicine sectors, being able to be used in the improvement of treatments and therapies in regenerative medicine of bone tissues.
Las proteínas morfogenéticas óseas (BMPs) son una familia de factores de crecimientos implicados en gran variedad de funciones durante el desarrollo y en la regeneración tisular [1]. Además del papel tan importante que ejercen en procesos no relacionados con el desarrollo óseo, las BMPs ejercen funciones únicas durante el desarrollo y regeneración del sistema esquelético [2], presentando algunas de ellas la capacidad exclusiva de inducir la formación de hueso ectópico cuando son implantadas en vertebrados adultos [3,4].Bone morphogenetic proteins (BMPs) are a family of growth factors involved in great variety of functions during development and in tissue regeneration [1]. In addition to the important role they play in processes, not related to bone development, BMPs exercise functions unique during the development and regeneration of the skeletal system [2], presenting some of them the exclusive ability to induce ectopic bone formation when implanted in vertebrates adults [3,4].
La reparación de defectos óseos y fracturas es uno de los mayores problemas clínicos y económicos en la sociedad actual, realizándose anualmente más de 3 millones de injertos óseos. A pesar de las buenas propiedades de los autoinjertos [5], la alta morbilidad asociada a esta; aproximación y la cantidad limitada de material que se puede obtener de cada donante, han propiciado el desarrollo de nuevos productos para la reparación del tejido óseo dañado o perdido [6-10].The repair of bone defects and fractures is one of the biggest clinical and economic problems in society current, performing more than 3 million bone grafts annually. Despite the good properties of autografts [5], the high morbidity associated with it; approach and the limited amount of material that can be obtained from each donor, have led to development of new products for bone tissue repair damaged or lost [6-10].
Las BMPs recombinantes deben ser utilizadas en combinación con un material transportador para favorecer su retención en el lugar del implante [11,12], lo que además permite que la liberación de la proteína al medio extracelular sea más lenta. Dos de los transportadores más utilizados en la actualidad son la matriz ósea desmineralizada (DBM) y el colágeno. La DBM presenta buenas propiedades para la retención y liberación de las BMPs [13]. De hecho, la DBM, por sí sola, tiene la capacidad de inducir la formación de hueso ectópico debido a la gran cantidad y variedad de factores de crecimiento que retiene tras la desmineralización [14, 15, 16], los efectos biológicos observados no pueden ser atribuidos únicamente a la proteína de fusión; además, otros estudio sugieren que la DBM contiene proteínas que pueden bloquear parcialmente la actividad de las BMPs [17]. Por otro lado, los efectos locales de los implantes de DBM son impredecibles, pudiendo incluso ser rechazados tras un periodo de tiempo, como ocurre frecuentemente con los aloinjertos cuando se usan en las cirugías óseas.Recombinant BMPs should be used in combination with a conveyor material to favor its retention at the implant site [11,12], which also allows that the release of the protein to the extracellular environment is more slow. Two of the most used transporters today they are demineralized bone matrix (DBM) and collagen. DBM It has good properties for retention and release of BMPs [13]. In fact, the DBM, by itself, has the ability to induce ectopic bone formation due to the large amount and variety of growth factors that you retain after demineralization [14,15,16], the biological effects observed not they can be attributed only to the fusion protein; also, other studies suggest that DBM contains proteins that can partially block the activity of BMPs [17]. On the other hand, the local effects of DBM implants are unpredictable, can even be rejected after a period of time, such as occurs frequently with allografts when used in bone surgeries
Por el contrario, el colágeno es considerado en la actualidad como uno de los mejores materiales osteoconductores, dada su versatilidad, alta biocompatibilidad y baja inmunogenicidad. El primer producto aprobado por la FDA compuesto por una BMP recombinante humana (rhBMP) para el tratamiento de fusiones espinales y fracturas tibiales abiertas, consiste en una combinación de esponja absorbible de colágeno (ACS), como matriz transportadora, y rhBMP-2 (INFUSE®, Medtronic, Minneapolis, MN, USA) o OP-1. Dado que la rhBMP-2 presenta baja afinidad natural por el colágeno, esta aproximación requiere el uso de altas dosis (miligramos) de BMPs [18, 19], lo cual no sólo puede tener efectos adversos colaterales, sino que eleva muchísimo el coste de estos tratamientos [20, 21].On the contrary, collagen is considered in Today as one of the best osteoconductive materials, given its versatility, high biocompatibility and low immunogenicity. The first FDA-approved product composed of a BMP recombinant human (rhBMP) for the treatment of fusions spinal and open tibial fractures, consists of a combination of collagen absorbable sponge (ACS), as a transport matrix, and rhBMP-2 (INFUSE®, Medtronic, Minneapolis, MN, USA) or OP-1. Since rhBMP-2 presents low natural affinity for collagen, this approach requires the use of high doses (milligrams) of BMPs [18, 19], which not only it can have adverse side effects, but it raises a lot the cost of these treatments [20, 21].
Gracias a la tecnología de ADN recombinante, se han modificado muchas proteínas de interés terapéutico, con el fin de favorecer su unión a diversos biomateriales [22-24] y reducir la pérdida de moléculas efectivas, ya sea por difusión o por degradación enzimática, manteniéndolas en el lugar de aplicación a la concentración farmacológica adecuada. Una de estas modificaciones consiste en la producción de una proteína de fusión constituida por el factor de crecimiento de interés y un dominio de unión a colágeno procedente del factor de von Willebrand (vWF). Se ha demostrado que esta aproximación aumenta de manera efectiva la capacidad de unión de algunas proteínas a colágeno [25-29].Thanks to recombinant DNA technology, it they have modified many proteins of therapeutic interest, in order to favor its union to various biomaterials [22-24] and reduce the loss of effective molecules, either by diffusion or by enzymatic degradation, keeping them in the place of application at the appropriate pharmacological concentration. One of these modifications consists in the production of a fusion protein constituted by the growth factor of interest and a collagen binding domain from the factor of von Willebrand (vWF). It has been shown that this approach effectively increases the binding capacity of some collagen proteins [25-29].
A pesar de que la producción de una rhBMP-2 modificada para favorecer su unión a colágeno ya ha sido abordada por otros autores [34, 35], todas esas proteínas han sido producidas con dominios adicionales para su purificación, y sus propiedades osteoinductoras sólo han sido demostradas en combinación con DBM. En particular, y en relación a secuencias adicionales que faciliten la purificación de proteínas, dichos dominios pueden inducir el desplegamiento de las proteínas a los que son añadidos, la pérdida de actividad de la proteína, y imposibilitar el uso de requerimientos para su aplicación terapéutica [37]. En particular, un dominio de histidinas (His-tag) puede alterar las características de unión de las proteínas recombinantes que lo incorporan [38, 39].Although the production of a modified rhBMP-2 to favor its binding to collagen has already been addressed by other authors [34, 35], all these proteins have been produced with additional domains for purification, and their osteoinductive properties have only been demonstrated in combination with DBM. In particular, and in relation to additional sequences that facilitate the purification of proteins, said domains can induce the deployment of the proteins to which they are added, the loss of activity of the protein, and make it impossible to use requirements for its therapeutic application [ 37]. In particular, a histidine domain ( His-tag ) can alter the binding characteristics of the recombinant proteins that incorporate it [38, 39].
La BMP-2 es uno de los factores de crecimientos osteoinductores más potentes y además, ejerce múltiples funciones en otros muchos tejidos distintos al hueso. Por otro lado, el colágeno es la proteína más abundante de la matriz extracelular animal y es el único material osteoconductor que ha sido autorizado para su uso clínico, en combinación con BMP-2, para la reparación ósea. Dada su baja afinidad por el colágeno, la BMP-2 debe ser utilizada a concentraciones muy por encima de los niveles fisiológicos, lo cual puede tener efectos adversos colaterales, como la reabsorción por parte de osteoclastos [20] o dar lugar a una osificación poco homogénea, lo cual tiene como consecuencia la pérdida de fuerza mecánica [21]. Una aproximación efectiva para aumentar y limitar la concentración local de proteína al lugar del implante, es aumentar la capacidad específica de unión de la BMP-2 al colágeno. En este sentido, algunos factores de crecimiento han sido ya modificados con dominios adicionales a unión al colágeno derivados del factor de von Willebrand [26, 28, 29, 33, 34], de colagenasa [22, 35], o de fibronectina [23, 36].BMP-2 is one of the factors of more powerful osteoinductive growths and also exerts multiple functions in many other tissues other than bone. By on the other hand, collagen is the most abundant protein in the matrix animal extracellular and is the only osteoconducting material that has been authorized for clinical use, in combination with BMP-2, for bone repair. Given his discharge affinity for collagen, BMP-2 must be used at concentrations well above levels physiological, which can have adverse side effects, such as resorption by osteoclasts [20] or lead to a little homogeneous ossification, which results in the loss of mechanical strength [21]. An effective approach to increase and limit the local protein concentration to the place of implant, is to increase the specific binding capacity of the BMP-2 to collagen. In this regard, some factors growth have already been modified with additional domains to Collagen binding derived from von Willebrand factor [26, 28, 29, 33, 34], collagenase [22, 35], or fibronectin [23, 36].
La presente invención comprende la producción y aplicación de una proteína de fusión que consiste en una rhBMP-2 modificada para unirse específicamente a colágeno, con el fin de mejorar la reparación y formación de hueso. La proteína rhBMP-2 modificada comprendida en la presente invención ha sido modificada únicamente con un dominio de unión a colágeno, derivado del factor de von Willebrand. Para evitar errores en la formación de puentes disulfuro durante el proceso de replegamiento in vitro, la Cys-7 que se encuentra en la secuencia original del decapéptido ha sido reemplazada por una metionina [26]. No se han añadido secuencias de purificación con el fin de evitar cualquier posible alteración en el factor de crecimiento como los ya comentado en el estado de la técnica [37, 38, 39].The present invention comprises the production and application of a fusion protein consisting of a rhBMP-2 modified to specifically bind to collagen, in order to improve bone formation and repair. The modified rhBMP-2 protein comprised in the present invention has been modified only with a collagen binding domain, derived from von Willebrand factor. To avoid errors in the formation of disulfide bridges during the in vitro refolding process, the Cys-7 found in the original decapeptide sequence has been replaced by a methionine [26]. No purification sequences have been added in order to avoid any possible alteration in the growth factor as already mentioned in the prior art [37, 38, 39].
Los resultados obtenidos evidencian que la proteína de fusión rhBMP-2-CBD presenta mayor afinidad por el colágeno, y demuestran que esa unión es muy estable durante un largo periodo de tiempo y retiene la actividad osteoinductiva, siendo capaz se inducir la formación de hueso nuevo incluso a concentraciones por debajo de las conocidas para la rhBMP-2 nativa. Además, en combinación con un transportador colagénico libre de ningún otro factor de crecimiento, como una esponja absorbible de colágeno, material aprobado por la FDA para su uso clínico en la inducción de hueso ectópico, bajas concentraciones de rhBMP-2-CBD son capaces de inducir la formación de hueso nuevo cuando es implantada in vivo.The results obtained show that the rhBMP-2-CBD fusion protein has a higher affinity for collagen, and demonstrates that this binding is very stable for a long period of time and retains osteoinductive activity, being able to induce the formation of new bone. even at concentrations below those known for native rhBMP-2. In addition, in combination with a collagen transporter free of any other growth factor, such as a collagen absorbable sponge, FDA approved material for clinical use in induction of ectopic bone, low concentrations of rhBMP-2-CBD are capable of induce the formation of new bone when implanted in vivo .
Por todo ello, la presente invención supone una alternativa mejor y más segura al uso de rhBMP-2 nativa en su aplicación en medicina regenerativa de tejido óseo. De este modo, la proteína de fusión objeto de la misma (rhBMP-2-CBD) sería una buena candidata para su uso clínico, dado que podría utilizarse a menor concentración ya que permanecería retenida en el lugar de aplicación de manera más eficiente, y no sólo induce la formación de más hueso sino que este hueso es más maduro, según la cantidad y tamaño de las trabéculas, cavidades medulares y vasos sanguíneos observados; además, permite la producción de hueso nuevo utilizando concentraciones de proteína por debajo de la menor concentración osteoinductiva (460 ng) establecida por Wang y col. [9]. Por último, la formación de hueso nuevo, con osteocitos embebidos en una matriz mineralizada compacta, muestra que la rhBMP-2-CBD mantiene la capacidad osteoinductora de la BMP-2 nativa cuando se combina con otros materiales osteoconductores distintos al colágeno, como es la hidroxiapatita (HA).Therefore, the present invention implies a better and safer alternative to using rhBMP-2 native in its application in regenerative medicine of bone tissue. From this way, the fusion protein object of the same (rhBMP-2-CBD) would be a good candidate for clinical use, since it could be used at a lower concentration as it would remain retained at the place of application more efficiently, and not only induces the formation of more bone but this bone is more mature, depending on the quantity and size of the trabeculae, spinal cavities and blood vessels observed; In addition, it allows the production of new bone using protein concentrations below the lowest concentration osteoinductive (460 ng) established by Wang et al. [9]. By last, new bone formation, with osteocytes embedded in a matrix mineralized compact, shows that the rhBMP-2-CBD maintains capacity osteoinductor of the native BMP-2 when combined with other osteoconductive materials other than collagen, such as hydroxyapatite (HA).
Figura 1.- Representación esquemática de la proteína de fusión rhBMP-2-CBD. El dominio de unión a colágeno constituido por el decapéptido WREPSFMALS es añadido al extremo N terminal de una proteína BMP-2 madura. La cisterna original es sustituida por una metionina. Una glicina adicional actúa como nexo.Figure 1.- Schematic representation of the rhBMP-2-CBD fusion protein. He collagen binding domain constituted by the decapeptide WREPSFMALS is added to the N-terminal end of a protein BMP-2 matures. The original cistern is replaced by a methionine An additional glycine acts as a nexus.
Figura 2.- Análisis de la producción, renaturalización y purificación de la proteína de fusión rhBMP-2-CBD: (A) SDS-PAGE con tinción azul de Coomassie. Línea 1, contenido proteína celular total de E. coli transformada con el vector pET17b-BMP-2-CBD, después de 4 h de inducción con 1 mM IPTG. Línea 2, proteínas insolubles y cuerpos de inclusión recuperados tras lavar en presencia de Tritón X-100. Línea 3, proteína rhBMP-2-CBD tras 72 h del replegamiento in vitro. Línea 4, fracción libre de cromatografía de heparín-sefarosa. Línea 5, dímeros de rhBMP-2-CBD purificados mediante cromatografía de heparín-sefarosa. (B) Western blot con un anticuerpo anti-BMP-2. Línea 1, rhBMP-2 producida por células CHO (R&D Systems). Línea 2, dímeros purificados de rhBMP-2-CBD no reducida. Línea 3, rhBMP-2-CBD reducida.Figure 2.- Analysis of the production, renaturation and purification of the rhBMP-2-CBD fusion protein: (A) SDS-PAGE with Coomassie blue staining. Line 1, total cell protein content of E. coli transformed with the vector pET17b-BMP-2-CBD, after 4 h induction with 1 mM IPTG. Line 2, insoluble proteins and inclusion bodies recovered after washing in the presence of Triton X-100. Line 3, rhBMP-2-CBD protein after 72 h of refolding in vitro . Line 4, free fraction of heparin-sepharose chromatography. Line 5, rhBMP-2-CBD dimers purified by heparin-sepharose chromatography. (B) Western blot with an anti-BMP-2 antibody. Line 1, rhBMP-2 produced by CHO cells (R&D Systems). Line 2, purified dimers of rhBMP-2-CBD not reduced. Line 3, rhBMP-2-CBD reduced.
Figura 3.- Propiedades de unión a colágeno de la proteína rhBMP-2-CBD: discos de colágeno son incubado con diferentes concentraciones de rhBMP-2 o rhBMP-2-CBD. Las moléculas unidas son detectadas usando un anticuerpo anti-BMP-2 después de 2 h (A) ó 7 días (B) lavando con PBST. Análisis densitométrico. Los datos representados son media ± SD, usando una escala relativa (0-255) y n = 5; * p<0,001 frente a rhBMP-2.Figure 3.- Collagen binding properties of the rhBMP-2-CBD protein: discs collagen are incubated with different concentrations of rhBMP-2 or rhBMP-2-CBD. Bound molecules are detected using an antibody anti-BMP-2 after 2 h (A) or 7 days (B) washing with PBST. Densitometric analysis. The data represented are mean ± SD, using a relative scale (0-255) and n = 5; * p <0.001 versus rhBMP-2.
Figura 4.- Actividad ALP inducida por rhBMP-2 y rhBMP-2-CBD en mioblastos de ratón C2C12. Las células son crecidas durante 72 h con 0, 50, 100, 200, 300, 500 ó 1000 ng/ml de cada factor de crecimiento. Los datos representados son media \pm SD y n = 5; * p<0,01.Figure 4.- ALP activity induced by rhBMP-2 and rhBMP-2-CBD in mouse myoblasts C2C12. The cells are grown for 72 h with 0, 50, 100, 200, 300, 500 or 1000 ng / ml of each growth factor. The data represented are mean ± SD and n = 5; * p <0.01.
Figura 5.- Formación de hueso ectópico in vivo: (A, B) esponjas de colágeno con 0,5 y 10 microgramos de rhBMP-2-CBD, respectivamente. (C, D) esponjas de colágeno con 0,5 y 10 microgramos de rhBMP-2, respectivamente. (E, F) implantes de HA con 10 microgramos rhBMP-2-CBD y 10 microgramos rhBMP-2, respectivamente. (G) implante de HA (control negativo). Asterisco: trabéculas de hueso maduro. Flechas: osteoblastos sintetizando nueva matriz osteoide. MC: células mesenquimáticas indeferenciadas. BM: médula ósea, v: vasos sanguíneos, m: músculo. HA: hidroxiapatita. Barra de escala = 100 \mum.Figure 5.- Ectopic bone formation in vivo : (A, B) collagen sponges with 0.5 and 10 micrograms of rhBMP-2-CBD, respectively. (C, D) collagen sponges with 0.5 and 10 micrograms of rhBMP-2, respectively. (E, F) HA implants with 10 micrograms rhBMP-2-CBD and 10 micrograms rhBMP-2, respectively. (G) HA implant (negative control). Asterisk: mature bone trabeculae. Arrows: osteoblasts synthesizing new osteoid matrix. MC: undefined mesenchymal cells. BM: bone marrow, v: blood vessels, m: muscle. HA: hydroxyapatite. Scale bar = 100 µm.
Figura 6.- Formación de hueso ectópico in vivo a concentraciones baja de proteína: (H) tinción de von Kossa de esponja de colágeno incubada con 400 ng de rhBMP-2-CBD, mostrando un nódulo mineralizado. (I) Petroleoquímica con anti-osteopontina y contratinción con hematoxilina del mismo implante, mostrando un núcleo de osteoblastos positivos embebidos en una abundante matriz osteoide. (J) A mayor aumento se pueden observar grupos de células expresando osteopontina (preosteoblastos), todavía indistinguibles morfológicamente respecto de las células indiferenciadas de alrededor. Flechas: preosteoblastos expresando osteopontina. Barra. de escala = 30 \mum en (H) e (I), 20 \mum en (J).Figure 6.- Ectopic bone formation in vivo at low protein concentrations: (H) Collagen sponge von Kossa staining incubated with 400 ng of rhBMP-2-CBD, showing a mineralized nodule. (I) Petrochemical with anti-osteopontin and hematoxylin contratinction of the same implant, showing a nucleus of positive osteoblasts embedded in an abundant osteoid matrix. (J) At higher magnification groups of cells expressing osteopontin (preosteoblasts), still morphologically indistinguishable from surrounding undifferentiated cells can be observed. Arrows: preosteoblasts expressing osteopontin. Bar. of scale = 30 µm in (H) and (I), 20 µm in (J).
A continuación se procede a detallar el modo de realización de la invención, que comprende la: obtención de la invención, su caracterización funcional, y la evaluación de su aplicabilidad.Then proceed to detail the mode of embodiment of the invention, comprising: obtaining the invention, its functional characterization, and the evaluation of its applicability.
Las células de osteosarcoma humana U-2 OS (HTB-96) se obtienen de la ATCC (American Type Culture Collection). Las células mioblasto de ratón C2C12 (91031101) se obtienen de la ECACC (European Collection of Cell Cultures). La BMP-2 recombinante humana (rhBMP-2) producida por células CHO es suministrada por R&D Systems (Minneapolis, MN, USA). El anticuerpo monoclonal anti-BMP-2 y el anti-IgG de ratón marcado con peroxidasa (HRP) son suministrados por Sigma-Aldrich (Steinheim, Germany). El anticuerpo monoclonal anti- osteopontina es obtenido del Banco de Hibridomas (Developmental Studies Hybridoma Bank, Iowa, IA, USA). El anticuerpo monoclonal anti-IgG de ratón biotinilado es suministrado por Abcam (Cambridge, MA, USA).Human osteosarcoma cells U-2 OS (HTB-96) are obtained from the ATCC (American Type Culture Collection). Myoblast cells C2C12 mouse (91031101) are obtained from the ECACC (European Collection of Cell Cultures). The recombinant human BMP-2 (rhBMP-2) produced by CHO cells is supplied by R&D Systems (Minneapolis, MN, USA). Monoclonal antibody anti-BMP-2 and the peroxidase-labeled mouse IgG (HRP) are supplied by Sigma-Aldrich (Steinheim, Germany). The monoclonal anti-osteopontin antibody is obtained of the Bank of Hybridomas (Developmental Studies Hybridoma Bank, Iowa, IA, USA). The mouse anti-mouse IgG monoclonal antibody Biotinylated is supplied by Abcam (Cambridge, MA, USA).
Se realiza la extracción del ARN total a partir de las células de osteosarcoma humano U-2 OS, las cuales se sabe que secretan BMPs [30]. El ADNc que codifica para la secuencia de la hBMP-2 se obtiene mediante RT-PCR utilizando oligonucleótidos específicos:Total RNA extraction is performed from of human osteosarcoma cells U-2 OS, the which are known to secrete BMPs [30]. The cDNA that codes for the sequence of the hBMP-2 is obtained by RT-PCR using specific oligonucleotides:
El ADNc obtenido se clona en el vector de
expresión pBIISK (+) (Stratagene, La Jolla, CA, USA). Los clones
seleccionados se utilizan para amplificar la secuencia que codifica
para el: dominio maduro de la BMP-2 humana, a la
cual se añade la secuencia del CBD en el extremo amino
(Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-Leu-Ser)
(figura 1). Para la rhBMP-2, el oligonucleónido
sentido es
5'-TTCATATGCAAGCCAAACACAAACAGC-3'.
Para la rhBMP-
2-CBD, es
5'-TTCATATGTGGCGCGAACCGAGCTTCATGGCTCTGAGCGGTGCAAGCCAAACACAAACAGC-3'.
En ambos casos, el oligonucleótido antisentido es
5'-AGAATTCCTGTACTAGCGACACCCAC-3'.
Ambos productos se clonan en el vector de expresión
pET-17b (EMD Biosciences/Merck Chemicals, Darmstadt,
Germany).The cDNA obtained is cloned into the pBIISK (+) expression vector (Stratagene, La Jolla, CA, USA). The selected clones are used to amplify the sequence encoding the: mature domain of human BMP-2, to which the CBD sequence is added at the amino terminus (Trp-Arg-Glu-Pro-Ser-Phe-Met -Ala-Leu-Ser) (figure 1). For rhBMP-2, the sense oligonucleonide is 5'-TTCATATGCAAGCCAAACACAAACAGC-3 '. For the rhBMP-
2-CBD, is 5'-TTCATATGTGGCGCGAACCGAGCTTCATGGCTCTGAGCGGTGCAAGCCAAACACAAACAGC-3 '. In both cases, the antisense oligonucleotide is 5'-AGAATTCCTGTACTAGCGACACCCAC-3 '. Both products are cloned into the pET-17b expression vector (EMD Biosciences / Merck Chemicals, Darmstadt, Germany).
Las proteínas recombinantes rhBMP-2 y rhBMP-2-CBD se expresan en E. coli, se repliegan in vitro, y se purifican según el método previamente descrito [31, 32] con ligeras modificaciones. Brevemente, las construcciones obtenidas pET-17b-BMP-2 y pET-17b-BMP-2-CBD se utilizan para transformar células de la cepa de E. coli Rosetta^{TM} (DE3). Los cultivos se realizan a 37ºC y en agitación y la inducción de la expresión de proteína recombinante se realiza mediante la adición de 1 mM de IPTG. Tras 4 h de inducción, se recuperan las células mediante centrifugación y se aíslan los cuerpos de inclusión mediante homogenización, centrifugación y varios lavados en presencia de Tritón X-100. Los cuerpos de inclusión obtenidos se solubilizan con 6 M de guanidinio hidrocloruro (Gnd-HCl), y las proteínas rhBMP-2 y rhBMP-2-CBD se repliegan durante 72 h a 10ºC en presencia de Gnd-HCl, ácido 2-(cyclohexylamino)ethanosulfonico (CHES) y glutation reducido:oxidado (GSH:GSSG) en una proporción 2:1. Tras el replegamiento, las proteínas diméricas obtenidas rhBMP-2 y rhBMP-2-CBD se purifican mediante cromatografía de afinidad por heparina, utilizando columnas de heparin-sefarosa HiTrap^{TM} (GE Healthcare, Uppsala, Sweden).The recombinant rhBMP-2 and rhBMP-2-CBD proteins are expressed in E. coli , replicated in vitro , and purified according to the previously described method [31, 32] with slight modifications. Briefly, the constructs obtained pET-17b-BMP-2 and pET-17b-BMP-2-CBD are used to transform cells of the E. coli Rosetta? Strain (DE3). Cultures are performed at 37 ° C and under stirring and induction of recombinant protein expression is performed by adding 1 mM of IPTG. After 4 h induction, the cells are recovered by centrifugation and the inclusion bodies are isolated by homogenization, centrifugation and several washes in the presence of Triton X-100. The inclusion bodies obtained are solubilized with 6 M guanidinium hydrochloride (Gnd-HCl), and the rhBMP-2 and rhBMP-2-CBD proteins are retracted for 72 h at 10 ° C in the presence of Gnd-HCl, acid 2- (cyclohexylamino) ethanosulfonic (CHES) and reduced glutathione: oxidized (GSH: GSSG) in a 2: 1 ratio. After refolding, the dimeric proteins obtained rhBMP-2 and rhBMP-2-CBD are purified by heparin affinity chromatography, using HiTrap? Heparin-sepharose columns (GE Healthcare, Uppsala, Sweden).
La adición de 1 mM de IPTG a los cultivos de células E. coli Rosetta (DE3) transformadas con la construcción pET-17b-BMP-2-CBD, favorece un alto rendimiento en la expresión de una proteína insoluble de unos \sim15 Kda, correspondiente al peso molecular esperado para los monómeros de rhBMP-2-CBD. El lavado de los extractos bacterianos con Tritón X-100 permite la obtención de la fracción insoluble de proteína constituida mayoritariamente por la forma de \sim15 KDa, la cual se encuentra en forma de cuerpos de inclusión. Tras la solubilización de las proteínas con Gnd-HCl, se realiza el plegamiento de las mismas in vitro durante 72 h, lo que da lugar a los dímeros de rhBMP-2-CBD, con una eficiencia de 40%. Finalmente los homodímeros de rhBMP-2-CBD, de unos \sim29 KDa, se purifican mediante cromatografía de afinidad por heparina, separándolos de los monómeros no dimerizados y del resto de proteínas contaminantes (figura 2A). El análisis mediante electroforesis y Western blot bajo condiciones reductoras y no-reductoras, usando un anticuerpo monoclonal anti-BMP-2, confirma que la proteína purificada es rhBMP-2-CBD (figura 2B). Mediante este método de producción el rendimiento obtenido es de 30 mg de rhBMP-2-CBD dimérica pura por litro de cultivo bacteriano. Resultados parecidos se obtienen en el caso de la producción, plegamiento y purificación de rhBMP-2 (datos no mostrados).The addition of 1 mM of IPTG to cultures of E. coli Rosetta (DE3) cells transformed with the construction pET-17b-BMP-2-CBD, favors a high yield in the expression of an insoluble protein of about \15 Kda, corresponding to the expected molecular weight for the rhBMP-2-CBD monomers. The washing of the bacterial extracts with Triton X-100 allows to obtain the insoluble fraction of protein constituted mainly by the form of ~ 15 KDa, which is in the form of inclusion bodies. After solubilization of the proteins with Gnd-HCl, folding them is performed in vitro for 72 h, which gives rise to the dimers of rhBMP-2-CBD, with an efficiency of 40%. Finally, the homodimers of rhBMP-2-CBD, of about ~ 29 KDa, are purified by heparin affinity chromatography, separating them from the dimerized monomers and the rest of contaminating proteins (Figure 2A). Analysis by electrophoresis and Western blotting under reducing and non-reducing conditions, using an anti-BMP-2 monoclonal antibody, confirms that the purified protein is rhBMP-2-CBD (Figure 2B). Through this production method the yield obtained is 30 mg of pure dimeric rhBMP-2-CBD per liter of bacterial culture. Similar results are obtained in the case of the production, folding and purification of rhBMP-2 (data not shown).
La afinidad de la rhBMP-2 y de la rhBMP-2-CBD por el colágeno tipo I se analiza in vitro, estudiando la capacidad de unión a ACS mediante el método de Kitajima y col. [23] ligeramente modificado. Para ello, se utiliza esponja absorbible de colágeno (derivada de piel bovina, cedida por el Dr. ME Nimni; patente US 5374539), que es cortada en discos de 5 mm de diámetro y 1 mm de grosor, y se lava con PBST (tampón fosfato salino con 0,1% de Tween 20). A cada disco se añaden 50 \mul de muestra y se incuba a 37ºC durante 2 h. Transcurrido el periodo de incubación las esponjas de colágeno se lavan con PBST y se inmuno tiñen con anti-BMP-2 y anti-IgG-HRP de ratón. La detección se realiza con el reactivo ECL (GE Healthcare). La cantidad de proteína unida a las esponjas de colágeno, se determina mediante densitometría, cuantificando la inmunorreactividad de las esponjas, utilizando el programa de sofware libre Image J (Rasband, WS., Image J, U.S. National Institute of Health, Bethesda, Maryland. USA) versión 1.38x. Para estudiar la estabilidad de la unión de la rhBMP-2-CBD, tras incubar las proteínas recombinantes durante 2 h a 37ºC, las esponjas de colágeno se lavan en PBST durante 7 días antes de realizar la inmunotinción con anticuerpos.The affinity of rhBMP-2 and rhBMP-2-CBD for type I collagen is analyzed in vitro , studying ACS binding capacity by the method of Kitajima et al. [23] slightly modified. For this, collagen absorbable sponge is used (derived from bovine skin, provided by Dr. ME Nimni; US patent 5374539), which is cut into discs 5 mm in diameter and 1 mm thick, and washed with PBST ( phosphate buffered saline with 0.1% Tween 20). 50 µl of sample are added to each disc and incubated at 37 ° C for 2 h. After the incubation period, the collagen sponges are washed with PBST and immuno stained with anti-BMP-2 and anti-mouse IgG-HRP. Detection is performed with the ECL reagent (GE Healthcare). The amount of protein bound to the collagen sponges is determined by densitometry, quantifying the immunoreactivity of the sponges, using the free software program Image J (Rasband, WS., Image J, US National Institute of Health, Bethesda, Maryland. USA) version 1.38x. To study the stability of the rhBMP-2-CBD binding, after incubating the recombinant proteins for 2 h at 37 ° C, the collagen sponges are washed in PBST for 7 days before performing immunostaining with antibodies.
En relación a la capacidad de unión a colágeno, mucha más cantidad de proteína rhBMP-2-CBD que rhBMP-2 permanece unida a las esponjas tras el lavado de 2 h en PBST (figura 3A). En lo que respecta a la estabilidad de la unión de ambas proteínas, en el caso de la rhBMP-2-CBD casi toda la proteína permanece unida a la esponja tras 7 días de lavado con PBST (figura 3B). Por el contrario, en el caso de la rhBMP-2, apenas queda proteína retenida en las esponjas tras este extenso lavado. Estos resultados demuestran que la rhBMP-2-CBD presenta mayor afinidad por el colágeno, siendo esta afinidad de más del doble que la proteína nativa a su mayor concentración (200 ng), y estable incluso después de un largo periodo de lavados.In relation to collagen binding capacity, much more protein rhBMP-2-CBD that rhBMP-2 remains attached to the sponges after the 2 h wash in PBST (figure 3A). In regards to the binding stability of both proteins, in the case of rhBMP-2-CBD almost all protein remains attached to the sponge after 7 days of washing with PBST (figure 3B). On the contrary, in the case of rhBMP-2, there is hardly any protein retained in the sponges after this extensive washed. These results demonstrate that the rhBMP-2-CBD has higher affinity for collagen, this affinity being more than double that of native protein at its highest concentration (200 ng), and stable even after a long period of washing.
La actividad biológica de las proteínas producidas rhBMP-2-CBD y rhBMP-2 se analiza in vitro mediante el ensayo de inducción de la actividad ALP en células C2C12. Estas células tienen la capacidad de diferenciarse hacia el linaje osteogénico en presencia de BMP-2. Las células mioblasto de ratón C2C12 se cultivan en DMEM suplementado con suero fetal de ternera 10% (v/v) y glutamina 2 mM, a 37ºC y en atmósfera de CO_{2} (condiciones estándar). Para realizar el ensayo de fosfatasa alcalina (ALP), las células se siembran en una placa de 96 pocillos (Nunclon, Nunc, Roskilde, Denmark) a una densidad de 3x10^{4} células/pocillo en medio DMEM con 2% FBS, 2 mM L-glutamine y rhBMP-2 o rhBMP-2-CBD. Como control se utiliza proteína rhBMP-2 producida por células CHO. Tras 72 horas de cultivo, las células se lavan con PBS y la actividad ALP se mide utilizando el método del p-nitrofenil fosfato (pNPP) (Sigma Fast^{TM} p-nitrophenyl phosphate tablets, Sigma-Aldrich).The biological activity of the proteins produced rhBMP-2-CBD and rhBMP-2 is analyzed in vitro by induction assay of ALP activity in C2C12 cells. These cells have the ability to differentiate into the osteogenic lineage in the presence of BMP-2. C2C12 mouse myoblast cells are cultured in DMEM supplemented with 10% fetal calf serum (v / v) and 2 mM glutamine, at 37 ° C and under CO 2 atmosphere (standard conditions). To perform the alkaline phosphatase (ALP) assay, the cells are seeded in a 96-well plate (Nunclon, Nunc, Roskilde, Denmark) at a density of 3x10 4 cells / well in DMEM medium with 2% FBS, 2 mM L-glutamine and rhBMP-2 or rhBMP-2-CBD. As a control, rhBMP-2 protein produced by CHO cells is used. After 72 hours of culture, the cells are washed with PBS and the ALP activity is measured using the p-nitrophenyl phosphate (pNPP) method (Sigma Fast ™ p-nitrophenyl phosphate tablets, Sigma-Aldrich).
La proteína de fusión rhBMP-2-CBD es capaz de inducir la actividad ALP en este tipo celular, de forma dosis dependiente, alcanzando valores similares a los obtenidos cuando se aplican las proteínas sin modificar rhBMP-2 producida en E. coli o producida por las células CHO (figura 4). No se observan diferencias significativas en ninguna de las concentraciones estudiadas. De acuerdo con estos resultados, se puede concluir que la adición del dominio de unión al colágeno no afecta a la actividad biológica de la proteína in vitro.The rhBMP-2-CBD fusion protein is capable of inducing ALP activity in this cell type, in a dose-dependent manner, reaching similar values to those obtained when unmodified rhBMP-2 proteins produced in E. coli or produced by CHO cells (figure 4). There are no significant differences in any of the concentrations studied. Based on these results, it can be concluded that the addition of the collagen binding domain does not affect the biological activity of the protein in vitro .
La actividad osteoinductora de la proteína de fusión producida, se analiza mediante el ensayo de inducción de hueso ectópico en rata, implantando junto al músculo dorsal, esponjas de colágeno incubadas con diferentes cantidades de rhBMP-2-CBD o rhBMP-2. El cuidado de los animales y los procedimientos utilizados, se realizan de acuerdo a la normativa internacional. En este estudio se utilizan ratas Wistar jóvenes (12 semanas). La ACS es cortada en disco de 5 mm de diámetro y 1 mm de grosor, que se incuban durante 2 h a 37ºC con soluciones de proteína que contienen 0,4, 0,5, 10 \mug rhBMP-2-CBD, ó 0,4, 0,5, 10 \mug rhBMP-2. Los controles negativos se incubaron con solución vehículo. Se realizan tres réplicas de cada una de las condiciones. Los animales son anestesiados, se desinfecta la región con solución de etanol al 70% y se procede a afeitar la piel de la región doral. Tras realizar una incisión en la zona media doral se colocan tres implantes con proteína en un lado del músculo dorsal y tres implantes control en el músculo contralateral. En otro grupo experimental, se realizan implantes con HA (ProOsteon 500®) impregnadas con 10 \mug de rhBMP-2-CBD o rhBMP-2. Tras 28 días de implante, los animales son sacrificados y los implantes extraídos y disecados del tejido circundante. El material extraído se fija con formaldehido al 4% durante 18 h y se procesa para su inclusión en parafina. Se realizan secciones transversales de 10 \mum de grosor que se tiñen con hematoxilina-eosina (H-E). Los implantes que contenían 0,4 \mug de proteína se estudian, además, mediante tinción de von Kossa e inmunohistoquímica, utilizando un anticuerpo monoclonal anti-osteopontina y un anti-IgG de ratón marcado con biotina.The osteoinductive activity of protein fusion produced, is analyzed by the induction test of ectopic bone in rat, implanted next to the dorsal muscle, collagen sponges incubated with different amounts of rhBMP-2-CBD or rhBMP-2. The care of animals and procedures used, are performed according to the regulations international. In this study young Wistar rats are used (12 weeks) The ACS is cut into a 5 mm diameter and 1 mm diameter disc. thickness, which are incubated for 2 h at 37 ° C with protein solutions containing 0.4, 0.5, 10 \ mug rhBMP-2-CBD, or 0.4, 0.5, 10 \ mug rhBMP-2. Negative controls were incubated with vehicle solution. Three replicas of each of the terms. The animals are anesthetized, the region is disinfected with 70% ethanol solution and proceed to shave the skin of the doral region After making an incision in the middle doral area, they place three implants with protein on one side of the dorsal muscle and three control implants in the contralateral muscle. In another group experimental, implants are performed with HA (ProOsteon 500®) impregnated with 10 \ mug of rhBMP-2-CBD or rhBMP-2. After 28 days of implant, the animals are sacrificed and implants removed and dissected from the tissue surrounding. The extracted material is fixed with 4% formaldehyde for 18 h and processed for inclusion in paraffin. Are made 10 µm thick cross sections that are stained with hematoxylin-eosin (H-E). The Implants containing 0.4 µg of protein are also studied by von Kossa staining and immunohistochemistry, using a monoclonal anti-osteopontin antibody and a anti-biotin labeled mouse IgG.
Tras cuatro semanas en el animal, los implantes extraídos aumentan de tamaño y presentan mayor dureza. El porcentaje de recuperación es del 100%, salvo en los controles negativos, implantados sin proteína recombinante, y las esponjas impregnadas con 0,4 \mug de rhBMP-2, que son reabsorbidos no encontrándose vestigio de material en el lugar de implantación. El estudio histológico realizado de las esponjas impregnadas con 0,5 y 10 \mug de rhBMP-2-CBD, teñidas con H-E, muestra la formación de hueso nuevo que se extiende homogéneamente por todo el implante, invasión de gran cantidad de vasos sanguíneos y generación de una médula ósea aparente. Se observan numerosos osteocitos embebidos en la matriz mineralizada de apariencia trabecular. En la periferia de la formación ectópica, se localizan células mesenquimáticas indiferenciadas y osteoblastos, en contacto directo con el nuevo hueso formado, sintetizando nueva matriz osteoide (figura 5, A y B). En el caso de la rhBMP-2 los implantes también muestran la formación de hueso nuevo, pero de apariencia claramente diferente al anterior, con una matriz más desorganizada y menos compacta, pocas cavidades medulares y menor angiogénesis con pocos vasos sanguíneos y de menor calibre. Las células mesenquimales indiferenciadas se localizan no sólo en la periferia del nuevo tejido formado, sino también en el interior del implante entre la poca matriz ósea desorganizada (figura 5, C y D). Todas estas evidencias sugieren que el hueso ectópico producido por la rhBMP-2 es más inmaduro que el producido por la rhBMP-2-CBD.After four weeks in the animal, the implants extracted increase in size and have greater hardness. The percentage recovery is 100%, except for negative controls, implanted without recombinant protein, and impregnated sponges with 0.4 µg of rhBMP-2, which are not reabsorbed finding trace of material in the place of implantation. He histological study of sponges impregnated with 0.5 and 10 \ mug of rhBMP-2-CBD, stained with H-E, it shows the formation of new bone that homogeneously extends throughout the implant, invasion of large amount of blood vessels and generation of a bone marrow apparent. Numerous osteocytes embedded in the matrix are observed mineralized trabecular appearance. On the periphery of the ectopic formation, mesenchymal cells are located undifferentiated and osteoblasts, in direct contact with the new bone formed, synthesizing new osteoid matrix (Figure 5, A and B). In the case of the rhBMP-2 implants also show the formation of new bone, but clearly appearance different from the previous one, with a more disorganized matrix and less compact, few spinal cavities and less angiogenesis with few blood vessels and smaller caliber. Mesenchymal cells undifferentiated are located not only on the periphery of the new formed tissue, but also inside the implant between the little disorganized bone matrix (figure 5, C and D). All these evidence suggests that the ectopic bone produced by the rhBMP-2 is more immature than that produced by the rhBMP-2-CBD.
Los implantes de HA, utilizados para comprobar la actividad de la rhBMP-2-CBD en combinación con un transportador no colagénico, teñidos con H-E, muestran la formación de hueso nuevo que tapiza las paredes de los poros de la HA, bastante compacto y con numerosos osteocitos embebidos en la matriz ósea, en asociación con un tejido medular. En este caso, no se observan diferencias entre la actividad osteoinductora de ambas proteínas en combinación con este transportador, lo cual demuestra que la adición del CBD no disminuye la actividad biológica de la BMP-2 cuando es combinada con otro material no colagénico (figura 5, E-G).HA implants, used to check the activity of the rhBMP-2-CBD in combination with a non-collagenic conveyor, stained with H-E, show the formation of new bone upholstery the pore walls of the HA, quite compact and with numerous osteocytes embedded in the bone matrix, in association with a tissue medullary. In this case, there are no differences between the osteoinductive activity of both proteins in combination with this transporter, which shows that the addition of CBD does not decrease the biological activity of BMP-2 when it is combined with other non-collagenic material (figure 5, E-G).
Los implantes con baja concentración de proteína, por debajo del límite efectivo, establecido por otros autores [9], sólo son recuperados en el caso de las esponjas de colágeno impregnadas con 400 ng de rhBMP-2-CBD. En este caso, la tinción con von Kossa pone de manifiesto la presencia de nódulos mineralizados de hueso nuevo en la región interna del implante (figura 6H). En estos nódulos, la inmunotinción con anti-osteopontina muestra grupos de osteoblastos que expresan este marcador temprano de la osteogénesis, embebidos entre abundante matriz osteoide (figura 6I). También pueden encontrarse pequeños grupos de preosteoblastos, entre las células mesenquimales indiferenciadas, característicos de la aparición de nuevos núcleos de osificación (figura 6J).Implants with low concentration of protein, below the effective limit, set by others authors [9], are only recovered in the case of sponges collagen impregnated with 400 ng of rhBMP-2-CBD. In this case, the Staining with von Kossa shows the presence of nodules mineralized from new bone in the internal region of the implant (figure 6H). In these nodules, immunostaining with anti-osteopontin shows groups of osteoblasts that express this early marker of osteogenesis, embedded between abundant osteoid matrix (figure 6I). Can also be found small groups of preosteoblasts, between mesenchymal cells undifferentiated, characteristic of the appearance of new nuclei of ossification (figure 6J).
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Claims (19)
- a.to.
- Extracción de ARN total a partir de células que secreten BMPs;Total RNA extraction from cells that secrete BMPs;
- b.b.
- Amplificación mediante RT-PCT usando los oligonucleótidos específicos 5'-TTTCTAGATGGACGCTCTTT CAATGGACG-3' y 5'-TTGGTACCCTAGCGACACCCACAACCCTCC-3' (sentido y antisentido, respectivamente);Amplification by RT-PCT using specific oligonucleotides 5'-TTTCTAGATGGACGCTCTTT CAATGGACG-3 'and 5'-TTGGTACCCTAGCGACACCCACAACCCTCC-3 ' (sense and antisense, respectively);
- c.C.
- Clonación del ADNc obtenido en un vector de expresión, transformación de E. coli, y selección de clones;Cloning of the cDNA obtained in an expression vector, transformation of E. coli , and selection of clones;
- d.d.
- Amplificación de la secuencia que codifica para el dominio maduro de la BMP-2, a la cual se añade la secuencia del CBD en el extremo amino, a partir de los clones seleccionados usando los oligonucleótidos específicos 5'-TTCATATGTGGCGCGAACCGAGCTTCATGGCTCTGAGCGGTGCAAGCCAAACACAAACAGC-3' y 5'-AGAATTCCTGTACTAGCGACACCCAC-3' (sentido y antisentido, respectivamente);Sequence amplification that encodes for the mature domain of BMP-2, to the which is added the CBD sequence at the amino end, from the clones selected using the specific oligonucleotides 5'-TTCATATGTGGCGCGAACCGAGCTTCATGGCTCTGAGCGGTGCAAGCCAAACACAAACAGC-3 ' and 5'-AGAATTCCTGTACTAGCGACACCCAC-3 ' (sense and antisense, respectively);
- e.and.
- Clonación del producto resultante en un vector de expresión, y expresión de la proteína rhBMP-2-CBD en E. coli;Cloning of the resulting product into an expression vector, and expression of the rhBMP-2-CBD protein in E. coli ;
- f.F.
- Aislamiento y solubilización de los cuerpos de inclusión, y replegamiento de las proteínas rhBMP-2-CBD;Isolation and solubilization of inclusion bodies, and protein refolding rhBMP-2-CBD;
- g.g.
- Purificación de rhBMP-2-CBD mediante cromatografía de afinidad por heparina.Purification of rhBMP-2-CBD by chromatography of affinity for heparin.
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| CHEN, B. et al. "{}Activation of demineralized bone matrix by genetically engineered human bone morphogenetic protein-2 with a collagen binding domain derived from von Willebrand factor propolypeptide"{}. JOURNAL OF BIOMEDICAL MATERIALS RESEARCH, PART A. 2007. Vol. 80A, N$^{o}$. 2, páginas 428-434; figura 1. * |
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