ES2257158B1 - PROCEDURE FOR THE PRODUCTION AND PURIFICATION OF SOMATOSTATIN IN ESCHERICHIA COLI CELLS FROM EXPRESSION IN THE SAME OF THE CODING GENE CLONED IN THE VECTOR PGRV1. - Google Patents
PROCEDURE FOR THE PRODUCTION AND PURIFICATION OF SOMATOSTATIN IN ESCHERICHIA COLI CELLS FROM EXPRESSION IN THE SAME OF THE CODING GENE CLONED IN THE VECTOR PGRV1. Download PDFInfo
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- ES2257158B1 ES2257158B1 ES200401780A ES200401780A ES2257158B1 ES 2257158 B1 ES2257158 B1 ES 2257158B1 ES 200401780 A ES200401780 A ES 200401780A ES 200401780 A ES200401780 A ES 200401780A ES 2257158 B1 ES2257158 B1 ES 2257158B1
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- plasmid
- somatostatin
- pgrv1
- baselineskip
- escherichia coli
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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Classifications
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- 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
- C07K14/575—Hormones
- C07K14/655—Somatostatins
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
Procedimiento para la producción y purificación de somatostatina en células de Escherichia coli a partir de la expresión en las mismas del gen codificante clonado en el vector pGRV1. Mediante técnicas básicas de ingeniería genética se ha clonado el extremo N-terminal de la beta-galactosidasa de Kluyveromyces lactis en el plásmido comercial pET17xb, generando dos sitios de restricción únicos susceptibles de ser utilizados para la clonación de secuencias codificantes de péptidos, dando lugar al plásmido pGRV1. La secuencia codificante de la hormona somatostatina se ha obtenido mediante una amplificación en cadena de la polimerasa (PCR) utilizando oligonucleótidos solapantes, clonando posteriormente el gen en el plásmido pGRV1, para dar lugar al plásmido pSOMA. La cepa de Escherichia coli BL21 (DE3) se transformó con el plásmido pSOMA dando lugar a la cepa CECT5840. Utilizando equipos de fermentación convencional se han obtenido cantidades suficientes de proteína de fusión, que tras su digestión con bromuro de cianógeno se han purificado cromatográficamente. El péptido obtenido muestra propiedades cromatográficas coincidentes con las de un patrón comercial.Procedure for the production and purification of somatostatin in Escherichia coli cells from the expression therein of the coding gene cloned in the vector pGRV1. Through basic genetic engineering techniques, the N-terminal end of the beta-galactosidase of Kluyveromyces lactis has been cloned in the commercial plasmid pET17xb, generating two unique restriction sites that can be used for cloning of peptide coding sequences, giving rise to plasmid pGRV1. The somatostatin hormone coding sequence has been obtained by polymerase chain amplification (PCR) using overlapping oligonucleotides, subsequently cloning the gene into plasmid pGRV1, to give rise to plasmid pSOMA. Escherichia coli strain BL21 (DE3) was transformed with plasmid pSOMA giving rise to strain CECT5840. Using conventional fermentation equipment, sufficient amounts of fusion protein have been obtained, which, after digestion with cyanogen bromide, have been chromatographically purified. The peptide obtained shows chromatographic properties coinciding with those of a commercial standard.
Description
Procedimiento para la producción y purificación de somatostatina en células de Escherichia coli a partir de la expresión en las mismas del gen codificante clonado en el vector pGRV1.Procedure for the production and purification of somatostatin in Escherichia coli cells from the expression therein of the coding gene cloned in the vector pGRV1.
La presente invención se refiere al proceso de producción biológica y posterior purificación de una preparación para uso farmacéutico de somatostatina humana.The present invention relates to the process of biological production and subsequent purification of a preparation for pharmaceutical use of human somatostatin.
La somatostatina (factor inhibitorio de la liberación de somatotropina) es un tetradecapéptido aislado inicialmente a partir del hipotálamo ovino por su capacidad para inhibir la liberación de la hormona del crecimiento de las células pituitarias anteriores (Brazeau P, Vale W, Burgus R, Ling N, Butcher M, Rivier J, Guillemin R. Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone (Science 1973: 179, 77-9). Posteriormente se ha demostrado su existencia en otros tejidos donde posee funciones muy diversas, entre las que cabe citar la modulación de la actividad neuronal y la secreción endocrina y exocrina.Somatostatin (inhibitory factor of somatotropin release) is an isolated tetradecapeptide initially from the sheep hypothalamus because of its ability to inhibit the release of growth hormone from cells anterior pituitaries (Brazeau P, Vale W, Burgus R, Ling N, Butcher M, Rivier J, Guillemin R. Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone (Science 1973: 179, 77-9). It has subsequently proven its existence in other tissues where it has very various, including the modulation of the activity Neural and endocrine and exocrine secretion.
Los efectos fisiológicos de esta hormona pueden agruparse en cuatro grandes grupos. Por una parte, la somatostatina actúa sobre la glándula pituitaria inhibiendo la liberación de una variedad de hormonas como la hormona del crecimiento, prolactina, glucagón, insulina, gastrina y hormonas estimulantes del tiroides (Wass J.A., en Endocrinology, ed, DeGrott, L.J. 1989: 1, 152-166).The physiological effects of this hormone can group into four large groups. On the one hand, somatostatin acts on the pituitary gland inhibiting the release of a variety of hormones such as growth hormone, prolactin, glucagon, insulin, gastrin and thyroid stimulating hormones (Wass J.A., in Endocrinology, ed, DeGrott, L.J. 1989: 1, 152-166).
Además, estudios farmacológicos han evidenciado que la somatostatina ejerce un efecto inhibitorio a nivel de la motilidad intestinal, secreción del ácido clorhídrico, pepsina y liberación de gastrina, secreción pancreática exocrina, liberación estimulada de secretina y CCK-pancreocimina, secreción basal y estimulada de glucagón e insulina. Esta diversidad de propiedades ha sido objeto de estudio de aplicaciones terapéuticas y se han obtenido resultados positivos en el control de las hemorragias gastrointestinales por ruptura de varices esofágicas, complementando a otras medidas (escleroterapia, cirugía, etc) (Reynaert, H. and Geerts, A. Pharmacological rationale for the use of somatostatin and analogues in portal hypertension. Aliment. Pharmacol. Ther. 2003: 18, 375-86).In addition, pharmacological studies have shown that somatostatin exerts an inhibitory effect at the level of intestinal motility, secretion of hydrochloric acid, pepsin and gastrin release, exocrine pancreatic secretion, release stimulated secretin and CCK-pancreocimine, basal and stimulated secretion of glucagon and insulin. This diversity of properties has been the subject of application study therapeutic and positive results have been obtained in the control of gastrointestinal bleeding due to varicose veins rupture esophageal, complementing other measures (sclerotherapy, surgery, etc.) (Reynaert, H. and Geerts, A. Pharmacological rationale for the use of somatostatin and analogues in portal hypertension. Food. Pharmacol Ther. 2003: 18, 375-86).
Por otra parte, la somatostatina se utiliza como adyuvante en el tratamiento de las fistulas pancreáticas secretoras (inhibiendo la colecistoquinina y la secretina).On the other hand, somatostatin is used as adjuvant in the treatment of secretory pancreatic fistulas (inhibiting cholecystokinin and secretin).
No menos importante es la actividad neuromodulatoria dentro del sistema nervioso central, donde tiene un efecto múltiple sobre la transmisión neuronal.No less important is the activity neuromodulatory within the central nervous system, where it has a multiple effect on neuronal transmission.
En general, tanto la somatostatina como sus análogos se utilizan a nivel clínico para tratar varias neoplasias, así como aquellas enfermedades donde se requiera inhibir la secreción de la hormona del crecimiento (gigantismo y acromegalia).In general, both somatostatin and its analogs are used clinically to treat several neoplasms, as well as those diseases where it is required to inhibit the growth hormone secretion (gigantism and acromegaly).
Se ha producido mediante técnicas de DNA recombinante un péptido de interés farmacológico (somatostatina) (Itakura, K, Hirose, T, Crea, R, Riggs, AD, Heyneker, HL, Bolivar, F, Boyer, HW. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 1977: 198, 1056-63). El gen que codifica dicho péptido se ha donado en un plásmido de expresión constitutiva diseñado a partir de un vector comercial, de manera que exprese una proteína híbrida. Esta proteína está formada por una secuencia polipeptídica de origen microbiano (\beta-galactosidasa de Kluyveromyces lactis) que representa la porción amino-terminal, y el péptido de interés farmacológico que representa la porción carboxi-terminal. Las dos porciones están separadas por una metionina.A peptide of pharmacological interest (somatostatin) (Itakura, K, Hirose, T, Crea, R, Riggs, AD, Heyneker, HL, Bolivar, F, Boyer, HW) has been produced by recombinant DNA techniques. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 1977: 198, 1056-63). The gene encoding said peptide has been donated in a constitutive expression plasmid designed from a commercial vector, so as to express a hybrid protein. This protein is formed by a polypeptide sequence of microbial origin (β-galactosidase of Kluyveromyces lactis ) that represents the amino-terminal portion, and the peptide of pharmacological interest that represents the carboxy-terminal portion. The two portions are separated by a methionine.
La separación de los dos péptidos que componen la proteína híbrida se produce mediante el tratamiento con bromuro de cianógeno (CNBr), un compuesto que escinde de forma específica una cadena polipeptídica con una alta especificidad al nivel del residuo de metionina, en presencia de ácido fórmico al 70% (Methods in Enzimology 1967: 11, 238).The separation of the two peptides that make up The hybrid protein is produced by bromide treatment cyanogen (CNBr), a compound that specifically cleaves a polypeptide chain with high specificity at the level of methionine residue, in the presence of 70% formic acid (Methods in Enzimology 1967: 11, 238).
La somatostatina es un tetradecapéptido cíclico que posee la estructura de la hormona hipotalámica que inhibe la liberación de la hormona de crecimiento humana. Su composición aminoacídica se obtuvo a partir de la publicada en la Real Farmacopea Española 2001, pp. 3466:Somatostatin is a cyclic tetradecapeptide which has the structure of the hypothalamic hormone that inhibits the release of human growth hormone. His composition amino acid was obtained from the one published in the Royal Spanish Pharmacopoeia 2001, pp. 3466:
La invención consta de varias etapas:The invention consists of several stages:
A partir de la secuencia aminoacídica se ha deducido la secuencia nucleotídica de la somatostatina, adaptándola a Escherichia coli para optimizar el uso de codones.From the amino acid sequence the nucleotide sequence of somatostatin has been deduced, adapting it to Escherichia coli to optimize the use of codons.
SEC. ID. N°: 1SEC. ID. N °: 1
- 5'-GCGGGCTGCAAAAACTTTTTTTGGAAAACCTTTACCAGCTGC-3'5'-GCGGGCTGCAAAAACTTTTTTTGGAAAACCTTTACCAGCTGC-3 '
Basándonos en esta secuencia hemos diseñado un par de oligonucleótidos específicos, SOM-D (SEC: ID. N°:2) y SOM-C (SEC: ID. N°:3),Based on this sequence we have designed a pair of specific oligonucleotides, SOM-D (SEC: ID. N °: 2) and SOM-C (SEC: ID. N °: 3),
(SEC: ID. N°: 2)(SEC: ID. No.: 2)
- 5'-CGATTATGGCGGGCTGCAAAAACTTTTTTTGGAAAACCTTTACCAGCTGCTAAG-3'5'-CGATT ATG GCGGGCTGCAAAAACTTTTTTTGGAAAACCTTTACCAGCTGC TAA G-3 '
(SEC: ID. N°: 3)(SEC: ID. No.: 3)
- 5'-CATGCTTAGCAGCTGGTAAAGGTTTTCCAAAAAAAGTTTTTGCAGCCCGCCATAAT-3'.5'-CATGC TTA GCAGCTGGTAAAGGTTTTCCAAAAAAAGTTTTTGCAGCCCGC CAT AAT-3 '.
Ambos oligonucleótidos incorporan un triplete codificante ATG que permite la posterior digestión de la proteína híbrida con CNBr en el extremo 5', y una señal de terminación de la transcripción TAA reconocida por E. coli en el extremo 3'.Both oligonucleotides incorporate an ATG coding triplet that allows the subsequent digestion of the hybrid protein with CNBr at the 5 'end, and a TAA transcription termination signal recognized by E. coli at the 3' end.
La amplificación de la secuencia de la somatostatina se realizó en un volumen de 25 \mul que contenía 2.5 \mul de tampón de reacción, 2.5 \mul de oligonucleótido SOM-D 4 \muM, 2.5 \mul de oligonucleótido SOM-C 4 \muM, 2.5 \mul de cada desoxirribonucleótido trifosfato, dATP, dCTP, dTTP y dGTP 2 mM, 1 \mul de MgCl_{2} 50 mM y 2 unidades de Vent Polimerasa (New England Biolabs). La reacción de amplificación fue llevada a cabo mediante un ciclo de desnaturalización del DNA a 94ºC durante 5 minutos, seguido de 30 ciclos de amplificación: 30 segundos a 94ºC, 30 segundos a 52ºC y 30 segundos a 72ºC. El producto obtenido se clonó en el vector pUC18 digerido con SmaI generándose el plásmido intermedio pUC18-SOMA. La secuenciación del fragmento donado permitió corroborar que la construcción se correspondía con el producto deseado. A partir de este molde, se realizó un segundo ciclo de PCR en las condiciones antes descritas pero substituyendo los oligonucleótidos SOM-D y SOM-C por SOM-5F (SEC: ID. N°: 4) y SOM-3R (SEQ. ID. N°: 5)The somatostatin sequence amplification was performed in a volume of 25 µl containing 2.5 µL of reaction buffer, 2.5 µL of 4 µM SOM-D oligonucleotide, 2.5 µL of 4 µM SOM-C oligonucleotide , 2.5 µl of each deoxyribonucleotide triphosphate, dATP, dCTP, dTTP and 2 mM dGTP, 1 µL of 50 mM MgCl2 and 2 Vent Polymerase units (New England Biolabs). The amplification reaction was carried out by a DNA denaturation cycle at 94 ° C for 5 minutes, followed by 30 amplification cycles: 30 seconds at 94 ° C, 30 seconds at 52 ° C and 30 seconds at 72 ° C. The product obtained was cloned into the vector pUC18 digested with Sma I generating the intermediate plasmid pUC18-SOMA. Sequencing of the donated fragment made it possible to confirm that the construction corresponded to the desired product. From this template, a second PCR cycle was performed under the conditions described above but replacing the SOM-D and SOM-C oligonucleotides with SOM-5F (SEQ: ID. N °: 4) and SOM-3R (SEQ. ID No.: 5)
SEQ. ID. N°: 4I KNOW THAT. ID. N °: 4
- 5'-CGAAATCGATTATGGCGGGC TGC-3'5'-CGAA ATCGAT TATGGCGGGC TGC-3 '
SEQ. ID. N°: 5I KNOW THAT. ID. N °: 5
- 5'-GTCCGCATGCTTAGCAGC TGGTA-3' 5'-GCATGC TTAGCAGC TGGTA GTCC-3 '
Ambos oligonucleótidos adicionan una diana única a la construcción para el corte con enzimas de restricción (ClaI en SOM-5F y Sph I en SOM-3R) que facilita su posterior donación en el vector pGRV1.Both oligonucleotides add a single target to the construct for restriction enzyme cutting ( Cla I in SOM-5F and Sph I in SOM-3R) that facilitates its subsequent donation in the vector pGRV1.
El plásmido comercial pET17xb (Seed, B. Nature 1987 329: 840) se utilizó como base para la construcción del vector de expresión constitutiva. El gen LAC4 (\beta-galactosidasa de Kluyveromyces lactis) se ha donado utilizando como molde el plásmido p347 (Rubio-Texeira, M, Castrillo, JI, Adam, AC, Ugalde, UO, Polaina, J., Yeast 1998: 14, 827-37) en las condiciones antes descritas pero substituyendo los oligonucleótidos SOM-D y SOM-C por LAC417-5 ( SEQ. ID. N°: 6) y LAC417-3 (SEQ. ID. N°: 7).Commercial plasmid pET17xb (Seed, B. Nature 1987 329: 840) was used as the basis for the construction of the constitutive expression vector. The LAC4 gene (β-galactosidase of Kluyveromyces lactis ) has been donated using the plasmid p347 as a template (Rubio-Texeira, M, Castrillo, JI, Adam, AC, Ugalde, UO, Polaina, J., Yeast 1998: 14, 827-37) under the conditions described above but replacing the SOM-D and SOM-C oligonucleotides with LAC417-5 (SEQ. ID. No.: 6) and LAC417-3 (SEQ. ID. No.: 7).
SEQ. ID. N°: 6I KNOW THAT. ID. N °: 6
- 5'-CAGGCTAGCCATATGTCTTGCCTTATTCC-3'5'-CAG GCTAGC CATATGTCTTGCCTTATTCC-3 '
SEQ. ID. N°: 7I KNOW THAT. ID. N °: 7
- 5'-ATTACTAGTGAGCTCTTATTCAAAAGCGAGATC-3'5'-ATTACTAGT GAGCTC TTATTCAAAAGCGAGATC-3 '
Ambos oligonucleótidos adicionan una diana única al amplificado para el corte con enzimas de restricción (NheI en pLAC417-5 y SacI en pLAC417-3), lo que facilita su posterior clonación en el vector pET17xb digerido con NheI/SacI. El vector final se denominó pGRV1. Este vector permite la producción estable de elevadas cantidades de una proteína híbrida, sin la necesidad de utilización de inductores químicos de coste elevado (como es el caso del IPTG). El plásmido pGRV1 (SEQ. ID. N°: 8) posee dos dianas únicas, ClaI y SphI, que permiten la clonación dirigida de la secuencia del péptido que se desee expresar (p.e. somatostatina). El diagrama que describe la construcción del vector se muestra en la figura 1.Both oligonucleotides add a single target to the amplified one for cutting with restriction enzymes ( Nhe I in pLAC417-5 and Sac I in pLAC417-3), which facilitates its subsequent cloning into the pET17xb vector digested with Nhe I / Sac I. final vector was named pGRV1. This vector allows the stable production of high amounts of a hybrid protein, without the need to use high-cost chemical inducers (as is the case of the IPTG). Plasmid pGRV1 (SEQ. ID. No.: 8) has two unique targets, Cla I and Sph I, which allow the directed cloning of the peptide sequence to be expressed (eg somatostatin). The diagram describing the construction of the vector is shown in Figure 1.
Se procedió a digerir el plásmido pUC18-SOMA con los enzimas de restricción ClaI y SphI y se purificó el fragmento codificante para la somatostatina en un gel de agarosa de bajo punto de fusión. El fragmento linearizado se clonó en el plásmido pGRV1 cortado con los enzimas ClaI y SphI, generándose el plásmido pSOMA. Este plásmido se utilizó para transformar la cepa de Escherichia coli BL21 (DE3), en adelante BL21 (DE3) SOMA, adecuada para la obtención de elevadas cantidades de la proteína de fusión. BL21 (DE3) es un lisógeno de un derivado del bacteriófago lambda D69, que contiene el gen de la RNA polimerasa del fago T7 bajo el control del promotor lacUV5 (Studier, FW, Moffatt, BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 1986: 189, 113-30, Patente española ES2024734). El plásmido resultante se ha depositado en la Colección Española de Cultivos Tipo. El número asignado fue el CECT5840 con fecha 30/10/03. El diagrama que describe la construcción del vector de expresión (pSOMA) se muestra en la figura 2.Plasmid pUC18-SOMA was digested with the restriction enzymes Cla I and Sph I and the somatostatin coding fragment was purified on a low melting agarose gel. The linearized fragment was cloned into plasmid pGRV1 cut with the enzymes Cla I and Sph I, generating plasmid pSOMA. This plasmid was used to transform the Escherichia coli strain BL21 (DE3), hereinafter BL21 (DE3) SOMA, suitable for obtaining high amounts of the fusion protein. BL21 (DE3) is a lysogen from a bacteriophage lambda D69 derivative, which contains the T7 phage RNA polymerase gene under the control of the lacUV5 promoter (Studier, FW, Moffatt, BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes J. Mol. Biol. 1986: 189, 113-30, Spanish patent ES2024734). The resulting plasmid has been deposited in the Spanish Type Culture Collection. The assigned number was CECT5840 dated 10/30/03. The diagram describing the construction of the expression vector (pSOMA) is shown in Figure 2.
Formulación del medio de cultivo. El medio de cultivo LB (g/L de cultivo) (Triptona, 10; extracto de levadura, 5; NaCl, 10) fue utilizado de manera rutinaria para el cultivo de BL21 (DE3) SOMA. La producción a gran escala en fermentador se realizó en medio SL con la siguiente composición (g/L de cultivo) (peptona bacteriológica, 26.8; extracto de levadura, 21.4; NaCl 8.5; MgSO_{4}\cdot7 H_{2}O, 0.86; K_{2}HPO_{4}, 5.4; NaH_{2}PO_{2}\cdot2 H_{2}O, 1.6; lactosa, 2), pH final 7.0. Para asegurar el mantenimiento del plásmido, se adiciona a los medios ampicilina a una concentración final de 50 \mug/ml.Formulation of the culture medium. The middle of LB culture (g / L culture) (Triptone, 10; yeast extract, 5; NaCl, 10) was used routinely for the culture of BL21 (DE3) SOMA. The large-scale fermenter production was carried out in SL medium with the following composition (g / L culture) (peptone bacteriological, 26.8; yeast extract, 21.4; NaCl 8.5; MgSO 4 • 7 H 2 O, 0.86; K 2 HPO 4, 5.4; NaH 2 PO 2 • 2 H 2 O, 1.6; lactose, 2), final pH 7.0. To ensure the maintenance of the plasmid, the ampicillin media at a final concentration of 50 µg / ml.
Realización del preinóculo y condiciones de fermentación. Se inocula una colonia BL21 (DE3) SOMA en un matraz con LB y se cultiva a 37ºC durante 8 horas en un agitador orbital. En ese estadio, se transfieren las células al biorreactor (3% v/v) y se incuban durante 18 h manteniendo las siguientes condiciones: presión parcial de oxígeno > 25%, temperatura = 37ºC, agitación > 350 rpm. Tras el periodo de cultivo por lotes, se inicia un proceso de fermentación en continuo durante 24-48 horas.Realization of the pre-circle and conditions of fermentation. A BL21 (DE3) SOMA colony is inoculated into a flask with LB and grown at 37 ° C for 8 hours on an orbital shaker. At that stage, the cells are transferred to the bioreactor (3% v / v) and They are incubated for 18 h maintaining the following conditions: partial oxygen pressure> 25%, temperature = 37 ° C, stirring > 350 rpm. After the batch culture period, a continuous fermentation process for 24-48 hours.
Las células obtenidas se recogían en una centrífuga Beckman a 7000 rpm durante 20 minutos a 4ºC, se lavaban tres veces en volúmenes equivalentes de agua destilada.The cells obtained were collected in a Beckman centrifuge at 7000 rpm for 20 minutes at 4 ° C, washed three times in equivalent volumes of distilled water.
La lisis celular se realizaba incubando las células en tampón Tris\cdotHCl 0.15 M, pH 8.5, Triton-X100 2% a 37ºC durante 18 h con agitación suave. Posteriormente, se centrifugaba a 7000 rpm durante 30 minutos a 4ºC y se lavaba tres veces en volúmenes equivalentes de agua destilada. Finalmente, los cuerpos de inclusión se repartían en alícuotas.Cell lysis was performed by incubating the cells in Tris buffer 0.15 M, pH 8.5, Triton-X100 2% at 37 ° C for 18 h with stirring soft. Subsequently, it was centrifuged at 7000 rpm for 30 minutes at 4 ° C and washed three times in equivalent volumes of distilled water. Finally, the inclusion bodies were distributed in aliquots
La lisis de los cuerpos de inclusión se realizaba resuspendiéndolos en NaOH 50 mM durante 10 minutos. A continuación se centrifugaba a 7000 rpm durante 30 minutos a temperatura ambiente, se recogía el sobrenadante, esencialmente constituido por proteína de fusión, y se repartía en alícuotas de 50 mL.The lysis of the inclusion bodies is Performed by resuspending them in 50 mM NaOH for 10 minutes. TO then it was centrifuged at 7000 rpm for 30 minutes at room temperature, the supernatant was collected, essentially constituted by fusion protein, and was distributed in aliquots of 50 mL
La ruptura de la proteína de fusión de realizaba mediante el tratamiento con bromuro de cianógeno. Este agente químico es capaz de romper el enlace peptídico entre dos aminoácidos (Met-X), siendo X cualquiera de los 20 aminoácidos esenciales. La reacción consiste en la mezcla de ácido fórmico al 70% y CNBr (200 mg/lote) a temperatura ambiente durante 18 horas. Tras la incubación, se añaden nueve volúmenes de agua destilada para parar la reacción y se liofiliza el producto. Los residuos generados se neutralizan mediante la adición de NaOH 10 M hasta que el pH sea superior a 7 y se desechan como residuos químicos halogenados (Current Protocols in Protein Science 2000: 11.4.1).The fusion protein breakdown performed by treatment with cyanogen bromide. This agent chemist is able to break the peptide bond between two amino acids (Met-X), where X is any of the 20 essential amino acids. The reaction consists of the acid mixture 70% formic and CNBr (200 mg / lot) at room temperature for 18 hours. After incubation, nine volumes of water are added distilled to stop the reaction and the product is lyophilized. The generated waste is neutralized by the addition of 10 M NaOH until the pH is greater than 7 and discarded as waste halogenated chemicals (Current Protocols in Protein Science 2000: 11.4.1).
El producto intermedio liofilizado se resuspende en fase móvil A (ácido cítrico 20 mM, citrato sódico 20 mM y urea 6 M a pH 3.5) y se cromatografía sobre una resina de intercambio catiónico SP-HP (Amersham Biosciences, Upsala, Suecia). La resina se equilibraba con fase móvil A (2 volúmenes) antes de su utilización y se lavaba con fase móvil A, en una cantidad correspondiente a alrededor de 1 volumen antes de la elución. El péptido somatostatina se eluye con un escalón de NaCl (150 mM) en fase móvil A. La somatostatina se eluye como un pico único separado del portador (región N-terminal de la \beta-galactosidasa). En la figura 3 se muestra un perfil cromatográfico típico.The freeze-dried intermediate is resuspended in mobile phase A (20 mM citric acid, 20 mM sodium citrate and urea 6 M at pH 3.5) and chromatography on an exchange resin SP-HP cationic (Amersham Biosciences, Uppsala, Sweden). The resin was balanced with mobile phase A (2 volumes) before use and washed with mobile phase A, in a amount corresponding to about 1 volume before the elution The somatostatin peptide is eluted with a NaCl step (150 mM) in mobile phase A. Somatostatin elutes as a peak single separate from the carrier (N-terminal region of β-galactosidase). Figure 3 shows a typical chromatographic profile.
Las fracciones que contienen el péptido, tras su análisis en SDS-PAGE se combinan y se cromatografían sobre una resina de fase reversa (Oasis HLB 60/30 \mum, Waters n° de catálogo 1436567) para eliminar restos de sales en la muestra. La resina se equilibra con 4 volúmenes de fase móvil A (ácido trifluoroacético (TFA) al 0.1% v/v). Las muestras, adicionadas de TFA a una concentración final del 0.1% (v/v), se inyectan en la columna y se eluyen en fase móvil B (acetonitrilo al 30%).The fractions containing the peptide, after analysis in SDS-PAGE are combined and Chromatograph on a reverse phase resin (Oasis HLB 60/30 um, Waters catalog No. 1436567) to remove traces of salts In the sample. The resin is balanced with 4 mobile phase volumes A (0.1% v / v trifluoroacetic acid (TFA)). The samples, added of TFA at a final concentration of 0.1% (v / v), it injected into the column and eluted in mobile phase B (acetonitrile at 30%)
La determinación cromatográfica (HPLC) a nivel cualitativo y cuantitativo se efectuó en un equipo Waters Millenium dotado de un detector dual a 214 y 280 nm, optándose por la primera longitud de onda para los análisis. Las cromatografías se efectuaron con una columna Spherisorb ODS2 (C 18) 5 \mum (300 mm x 0.45) Waters operada a flujo constante de 1 ml/min. Las fases móviles utilizadas fueron: A, 0.1% TFA en agua y B, 0.1% TFA en acetonitrilo siguiendo el siguiente perfil: t=0, 90% A - 10% B; t=10, 20% A - 80% B; t=15, 20% A - 80% B; t=6 0,0% A - 100% B. El perfil cromatográfico típico se muestra en la figura 4.Chromatographic determination (HPLC) at the level qualitative and quantitative was carried out in a Waters Millenium team equipped with a dual detector at 214 and 280 nm, opting for the first wavelength for analysis. The chromatographs are made with a Spherisorb ODS2 column (C 18) 5 µm (300 mm x 0.45) Waters operated at a constant flow of 1 ml / min. Phases mobile phones used were: A, 0.1% TFA in water and B, 0.1% TFA in acetonitrile following the following profile: t = 0.90% A - 10% B; t = 10, 20% A - 80% B; t = 15, 20% A - 80% B; t = 6 0.0% A - 100% B. The Typical chromatographic profile is shown in Figure 4.
La determinación de la masa molecular se realizó mediante la técnica de MALDI-TOF en un espectrómetro Voyager-DE-PRO de la casa Applied Biosystems. El péptido en solución se aplica sobre una placa porta-muestra y después de ser evaporado a sequedad se añade sobre la muestra un volumen igual de la matriz ácido alfa-ciano-4-hidroxicinámico. La determinación de las medidas se realizó en modo reflector, utilizando polaridad positiva. Los parámetros empleados son los siguientes: el voltaje de aceleración utilizado fue de 20000 V, con un retraso en el tiempo de extracción de 180 nseg, se contabilizaron un total de 600 disparos con el láser para cada espectro. En la figura 5 se muestra un espectro tipo.Molecular mass determination was performed using the MALDI-TOF technique in a Voyager-DE-PRO spectrometer of the Applied Biosystems house. The peptide in solution is applied on a sample holder and after being evaporated to dryness an equal volume of the matrix is added to the sample acid alpha-cyano-4-hydroxycinnamic. The measurements were determined in reflector mode, using positive polarity. The parameters used are the following: the acceleration voltage used was 20,000 V, with a delay in the extraction time of 180 nsec, is they counted a total of 600 shots with the laser for each spectrum. A type spectrum is shown in Figure 5.
CNBr: bromuro de cianógenoCNBr: cyanogen bromide
FPLC: cromatografía líquida de resolución rápidaFPLC: liquid chromatography resolution fast
HPLC: cromatografía líquida de alta eficaciaHPLC: high efficiency liquid chromatography
IPTG: isopropil-\beta-D-tiogalacto-piranósidoIPTG: isopropyl-? -D-thiogalacto-pyranoside
MALDI-TOF: espectrometría de masas: desorción/ionización por láser con ayuda de matriz-tiempo de vueloMALDI-TOF: spectrometry of masses: laser desorption / ionization with the help of matrix-time of flight
PAGE: electroforesis en gel de poliacrialamidaPAGE: gel electrophoresis polyacrylamide
PCR: reacción en cadena de la polimerasaPCR: polymerase chain reaction
SDS: dodecil sulfato sódicoSDS: sodium dodecyl sulfate
TFA: ácido trifluoroacéticoTFA: trifluoroacetic acid
TRIS: tris-hidroximetil-aminometanoTRIS: tris-hydroxymethyl-aminomethane
La figura 1 representa la estrategia seguida para la construcción del plásmido pGRV1. Este plásmido posee dos dianas únicas, ClaI y SphI, que se utilizan para donar los péptidos que se desee expresar. Ap: gen de resistencia a ampicilina; ori: origen de replicación.Figure 1 represents the strategy followed for the construction of plasmid pGRV1. This plasmid has two unique targets, Cla I and Sph I, which are used to donate the peptides that you wish to express. Ap: ampicillin resistance gene; ori: origin of replication.
La figura 2 representa:Figure 2 represents:
- a)to)
- la estrategia seguida para la construcción del vector pSOMA, a partir del plásmido pGRV1 linearizado con ClaI y SphI y de la somatostatina generada mediante una reacción en cadena de la polimerasa (PCR).the strategy followed for the construction of the pSOMA vector, from the plasmid pGRV1 linearized with Cla I and Sph I and the somatostatin generated by a polymerase chain reaction (PCR).
- b)b)
- un diagrama de la proteína de fusión obtenida, que incluye el extremo N-terminal de la \beta-galactosidasa de Kluyveromyces lactis y la somatostatina.a diagram of the fusion protein obtained, which includes the N-terminal end of the β-galactosidase of Kluyveromyces lactis and somatostatin.
La figura 3 es un perfil de elución en FPLC mostrando la purificación de la somatostatina a partir de digestiones con CNBr de la proteína de fusión. La flecha indica el pico correspondiente a la somatostatina.Figure 3 is an elution profile in FPLC showing the purification of somatostatin from CNBr digestions of the fusion protein. The arrow indicates the peak corresponding to somatostatin.
La figura 4 es el perfil de elución de la
somatostatina en HPLC de fase reversa C18 en un gradiente de
agua/acetoni-
trilo con TFA al 0.1% en ambas fases. El pico
de elución se ha marcado con una flecha. La cuantificación del
péptido se efectuó mediante comparación con un patrón externo
comercial (Sigma).Figure 4 is the elution profile of somatostatin in C18 reverse phase HPLC in a water / acetonic gradient.
trilo with 0.1% TFA in both phases. The elution peak has been marked with an arrow. The quantification of the peptide was carried out by comparison with a commercial external standard (Sigma).
La figura 5 muestra un espectro de masas de la somatostatina obtenido mediante espectrometría de masas MALDI-TOF. La señal correspondiente al peso molecular de la somatostatina se marca con una flecha.Figure 5 shows a mass spectrum of the somatostatin obtained by mass spectrometry MALDI-TOF. The signal corresponding to the weight Somatostatin molecular is marked with an arrow.
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Claims (6)
- a)to)
- diseño de un gen que codifica para la somatostatina humana (SEQ. ID. N°: 2) adaptado al uso de codones en Escherichia coli design of a gene that codes for human somatostatin (SEQ. ID. No.: 2) adapted to the use of codons in Escherichia coli
- b)b)
- construcción del plásmido pGRV1 (1), (SEQ.ID.N°: 8)construction of plasmid pGRV1 (1), (SEQ.ID.No .: 8)
- c)C)
- clonación del gen de la somatostatina en el plásmido pGRV1, para obtener el vector pSOMAcloning of the somatostatin gene in plasmid pGRV1, to obtain the vector pSOMA
- d)d)
- transformación de la cepa Escherichia coli BL21-DE3 con el plásmido pSOMA, para obtener la cepa CECT5840transformation of the Escherichia coli BL21-DE3 strain with the plasmid pSOMA, to obtain the strain CECT5840
- e)and)
- fermentación en medio SL, bajo condiciones controladas, de la cepa CECT5840fermentation in SL medium, low controlled conditions of strain CECT5840
- f)F)
- extracción de proteína, permeabilización de cuerpos de inclusión, y purificación cromatográfica en dos fases (1, intercambio catiónico y 2, fase reversa) para obtener el producto final (somatostatina)protein extraction, permeabilization of inclusion bodies, and purification two-phase chromatographic (1, cation exchange and 2, phase reverse) to obtain the final product (somatostatin)
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| Application Number | Priority Date | Filing Date | Title |
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| ES200401780A ES2257158B1 (en) | 2004-07-09 | 2004-07-09 | PROCEDURE FOR THE PRODUCTION AND PURIFICATION OF SOMATOSTATIN IN ESCHERICHIA COLI CELLS FROM EXPRESSION IN THE SAME OF THE CODING GENE CLONED IN THE VECTOR PGRV1. |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200401780A ES2257158B1 (en) | 2004-07-09 | 2004-07-09 | PROCEDURE FOR THE PRODUCTION AND PURIFICATION OF SOMATOSTATIN IN ESCHERICHIA COLI CELLS FROM EXPRESSION IN THE SAME OF THE CODING GENE CLONED IN THE VECTOR PGRV1. |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5583013A (en) * | 1977-11-08 | 1996-12-10 | Genentech, Inc. | Method and means for microbial polypeptide expression |
| EP0108045B1 (en) * | 1982-10-25 | 1988-12-07 | Monsanto Company | Reca promoter dependent polypeptide production |
| JPH02235899A (en) * | 1989-03-08 | 1990-09-18 | Fujisawa Pharmaceut Co Ltd | New fused protein of somatostatin and production thereof |
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| ITAKURA K. et al. "Expression in Escherichia coli of a chemically synthesized gene". 1977. Science. Vol. 198, páginas 1056-1063. * |
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