Academic literature on the topic 'ProteÃna recombinante'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the lists of relevant articles, books, theses, conference reports, and other scholarly sources on the topic 'ProteÃna recombinante.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Journal articles on the topic "ProteÃna recombinante"
1
Gibertoni, A. M., M. C. M. Gonçalves, M. F. S. Montassier, C. C. Fernandes, and H. J. Montassier. "CLONAGEM, EXPRESSÃO E CARACTERIZAÇÃO DA NUCLEOPROTEÍNA RECOMBINANTE DO VÍRUS DA BRONQUITE INFECCIOSA EM ESCHERICHIA COLI E EM PICHIA PASTORIS." Arquivos do Instituto Biológico 77, no. 1 (March 2010): 1–9. http://dx.doi.org/10.1590/1808-1657v77p0012010.
Full textAbstract:
RESUMO O gene da proteína de nucleocapsídeo (1.230 pb) da estirpe M41 do vírus da bronquite infecciosa (VBI) foi amplificado pelas reações de transcrição reversa e em cadeia da polimerase (RT-PCR) e clonado, em seguida, em dois sistemas; pET28a - Escherichia coli e pFLD -Pichia pastoris. Os produtos recombinantes construídos para expressão (pET28a-N ou pFLD-N) foram identificados por análises de PCR e de sequenciamento de nucleotídeos. Os clones transformantes da linhagem BL21 de E. coli e da linhagem GS115 de P. pastoris foram submetidos aos protocolos apropriados de indução. A expressão da proteína N de fusão com etiqueta de poli-histidina e com massa molecular de 54 kDa foi determinada pelas técnicas de SDS-PAGE e de Western blotting, confirmando-se que ambas proteínas N recombinantes apresentaram tamanhos e antigenicidade compatíveis com a proteína N nativa do próprio VBI. O sistema E. coli expressou uma quantidade relevante da proteína N recombinante, enquanto que o sistema P. pastoris produziu uma baixa recuperação dessa proteína recombinante. A proteína N recombinante gerada pelo sistema bacteriano foi purificada em resina de níquel-sepharose. O conjunto de resultados indica que o sistema de expressão constituído por pET28a – E. coli é mais efetivo para produzir a proteína N recombinante do VBI destinada ao uso como antígeno para detectar anticorpos anti-virais específicos em ensaios de imunodiagnóstico para essa infecção viral.
2
Kim, Yoo-Gon, Woo-Jong Lee, Chan-Hee Won, Yong-Hee Kim, Ji-Sun Yun, Min-Seon Hong, and Chul-Soo Shin. "A study on short-term stability of recombinant protein A." Analytical Science and Technology 24, no. 3 (June 25, 2011): 193–99. http://dx.doi.org/10.5806/ast.2011.24.3.193.
Full text
3
Peterson, A. A. "Accumulation of recombinant fusion protein — secretory analog of Ag85B and ESAT6 Mycobacterium tuberculosis proteins – in transgenic Lemna minor L. Plants." Biotechnologia acta 8, no. 5 (2015): 38–48. http://dx.doi.org/10.15407/biotech8.05.039.
Full text
4
Bobek, L. A., H. Tsai, and M. J. Levine. "Expression of Human Salivary Histatin and Cystatin/ Histatin Chimeric cDNAs in Escherichia coli." Critical Reviews in Oral Biology & Medicine 4, no. 3 (April 1993): 581–90. http://dx.doi.org/10.1177/10454411930040034501.
Full textAbstract:
We have previously constructed recombinants encoding the full-length and truncated forms of cystatin-SN and expressed these in the Escherichia coli expression system pGEX-2T, which expresses foreign sequences as fusion proteins with glutathione S-transferase (GST). Recombinant cystatins were produced and purified in large quantities. The full-length recombinant cystatin-SN exhibited comparable biological activity and secondary structure to natural cystatin, validating the use of the full-length and mutant recombinant proteins for structure-function studies of salivary molecules. In this study, we have expressed histatin-1 cDNA in the pGEX-3X vector and cystatin-SN/histatin-1 or cystatin-SN/histatin-3 chimeric cDNAs in the pGEX-2T vector. Gene splicing by overlap extension (SOE), a PCR-based method, was used for generating the chimeric cDNAs. Each construct was analyzed by DNA sequencing, which showed the correct junctions and reading frames between the GST/histatin-1 and the GST/cystatin/histatin cDNAs. Expression of histatin and cystatin/histatin chimeras was induced by IPTG and the production of the fusion proteins monitored by SDS-PAGE/Coomassie blue staining and in the case of the GST/cystatin/histatin fusion proteins, also by Western blot using anti-cystatin antibody. The results of these studies showed that we have successfully constructed recombinants encoding the individual and chimeric salivary molecules and efficiently expressed these in E. coli expression system pGEX. Purification and characterization of recombinant histatin and cystatin-histatin hybrid proteins are presently ongoing.
5
Nieto Clavijo, Carlos Alfonso, Nicolás Forero Baena, and María Helena Ramírez Hernández. "Diseño y producción de diversas proteínas fusión de la nicotinamida/nicotinato mononucleótido adenilil transferasa (NMNAT) de Plasmodium falciparum." Revista Colombiana de Química 46, no. 3 (September 1, 2017): 5–10. http://dx.doi.org/10.15446/rev.colomb.quim.v46n3.63492.
Full textAbstract:
Las proteínas recombinantes se han convertido en herramientas útiles en la investigación bioquímica. Sin embargo, durante su producción, aparecen cuerpos de inclusión (IB), debido, por un lado, a la alta expresión de proteína producida a partir de los vectores usados que poseen promotores de alta eficiencia y, por otro lado, a características propias de la proteína. Ahora bien, la nicotinamida/nicotinato mononucleótido adenililtransferasa (NMNAT) es una proteína central en la biosíntesis del NAD(H)+, molécula esencial en el metabolismo celular, y ha sido estudiada en parásitos protozoos. Para el estudio de la NMNAT de estos parásitos se ha recurrido a la expresión de su versión recombinante en E. coli, obteniéndose gran cantidad de proteína como IB. Con el fin de aumentar la solubilidad de la proteína, se clonó la secuencia codificante de la NMNAT de Plasmodium falciparum en diferentes vectores de expresión, se indujo la expresión de la proteína recombinante en E. coli BL21(DE3) y se analizó la solubilidad. La proteína fusión con mayor solubilidad fue purificada y evaluada enzimáticamente. La adición de la etiqueta MBP (proteína de unión a maltosa) a la PfNMNAT incrementó su solubilidad y permitió obtener una proteína funcional con una alta pureza.
6
Astuti, R. W., N. Wijayanti, and A. Haryanto. "Expression of Recombinant Fusion Protein from Local Isolate of Newcastle Disease Virus and Antibody Response to Recombinant Fusion Protein in Broiler Chickens Post-Vaccination." Journal of the Indonesian Tropical Animal Agriculture 45, no. 2 (May 15, 2020): 78–90. http://dx.doi.org/10.14710/jitaa.45.2.78-90.
Full textAbstract:
This research aimed to express and purify the recombinant Fusion (F) protein of Newcastle Disease Virus (NDV) from a local isolate in Galur, Kulon Progo, Indonesia (0663/04/2013) from recombinant vector plasmid pBT7-N-His F, and to study the antibody response in the broiler sera which were injected with pure recombinant F protein compared with treated broilers that were vaccinated with commercial inactive NDV vaccines and control broilers without vaccination. The results showed that the recombinant F protein of NDV was successfully expressed, purified and visualized by SDS-PAGE with Coomassie Brilliant Blue staining and Westernblotting methods as a specific recombinant F protein with a molecular weight of 28 kDa. The pure recombinant F protein then was injected into broilers to determine the antibody response in broiler serum. Indirect ELISA showed that the production of antibodies was high in F protein vaccinated groups in comparison with other treated and control groups. The recombinant F protein has potential to be developed as a recombinant vaccine candidate after truncating the 6x His-tag part to obtain higher antibody respond if compared with antibody production in broiler serum post vaccinated with some commercially available broiler vaccines.
7
Ferrari, Luca, and Stefan G. D. Rüdiger. "Recombinant production and purification of the human protein Tau." Protein Engineering, Design and Selection 31, no. 12 (December 1, 2018): 447–55. http://dx.doi.org/10.1093/protein/gzz010.
Full textAbstract:
Abstract Tau protein is a microtubule-stabilising protein whose aggregation is linked to Alzheimer’s Disease and other forms of dementia. Tau biology is at the heart of cytoskeletal dynamics and neurodegenerative mechanisms, making it a crucial protein to study. Tau purification, however, is challenging as Tau is disordered, which makes it difficult to produce in recombinant system and is degradation-prone. It is thus challenging to obtain pure and stable preparations of Tau. Here, we present a fast and robust protocol to purify Tau recombinantly in Escherichia coli. Our protocol allows purifying Tau either tag-less or FLAG-tagged at its N-terminus, and Tau fragments of interest. By exploiting a cleavable affinity tag and two anion exchange columns, we obtained Tau preparations of high purity, stable and suitable for in vitro studies, including aggregation experiments that resemble neurodegenerative processes.
8
Santos, Anderson K., and Rodrigo R. Resende. "PRODUÇÃO DE PROTEÍNAS RECOMBINANTES HUMANAS: Boa, Barata E Em Larga Escala." Nanocell News 2, no. 8 (February 24, 2015): n/a. http://dx.doi.org/10.15729/nanocellnews.2015.02.24.006.
Full text
9
Cuervo, Nancy Stella, Sophie Guillot, Natalia Romanenkova, Mariana Combiescu, André Aubert-Combiescu, Mohamed Seghier, Valérie Caro, Radu Crainic, and Francis Delpeyroux. "Genomic Features of Intertypic Recombinant Sabin Poliovirus Strains Excreted by Primary Vaccinees." Journal of Virology 75, no. 13 (July 1, 2001): 5740–51. http://dx.doi.org/10.1128/jvi.75.13.5740-5751.2001.
Full textAbstract:
ABSTRACT The trivalent oral poliomyelitis vaccine (OPV) contains three different poliovirus serotypes. It use therefore creates particularly favorable conditions for mixed infection of gut cells, and indeed intertypic vaccine-derived recombinants (VdRec) have been frequently found in patients with vaccine-associated paralytic poliomyelitis. Nevertheless, there have not been extensive searches for VdRec in healthy vaccinees following immunization with OPV. To determine the incidence of VdRec and their excretion kinetics in primary vaccinees, and to establish the general genomic features of the corresponding recombinant genomes, we characterized poliovirus isolates excreted by vaccinees following primary immunization with OPV. Isolates were collected from 67 children 2 to 60 days following vaccination. Recombinant strains were identified by multiple restriction fragment length polymorphism assays. The localization of junction sites in recombinant genomes was also determined. VdRec excreted by vaccinees were first detected 2 to 4 days after vaccination. The highest rate of recombinants was on day 14. The frequency of VdRec depends strongly on the serotype of the analyzed isolates (2, 53, and 79% of recombinant strains in the last-excreted type 1, 2, and 3 isolates, respectively). Particular associations of genomic segments were preferred in the recombinant genomes, and recombination junctions were found in the genomic region encoding the nonstructural proteins. Recombination junctions generally clustered in particular subgenomic regions that were dependent on the serotype of the isolate and/or on the associations of genomic segments in recombinants. Thus, VdRec are frequently excreted by vaccinees, and the poliovirus replication machinery requirements or selection factors appear to act in vivo to shape the features of the recombinant genomes.
10
Fisher, Adam C., Charles H. Haitjema, Cassandra Guarino, Eda Çelik, Christine E. Endicott, Craig A. Reading, Judith H. Merritt, A. Celeste Ptak, Sheng Zhang, and Matthew P. DeLisa. "Production of Secretory and Extracellular N-Linked Glycoproteins inEscherichia coli." Applied and Environmental Microbiology 77, no. 3 (December 3, 2010): 871–81. http://dx.doi.org/10.1128/aem.01901-10.
Full textAbstract:
ABSTRACTTheCampylobacter jejuni pglgene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred intoEscherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competentE. colicells. With this “glycosylation tag,” a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that theC. jejuniN-glycosylation machinery is compatible with distinct secretory mechanisms inE. coli, effectively expanding the N-linked glycome of recombinantE. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates.
Dissertations / Theses on the topic "ProteÃna recombinante"
1
Oliveira, Raquel Sombra BasÃlio de. "ExpressÃo heterÃloga, caracterizaÃÃo cristalogrÃfica e anÃlise funcional de uma osmotina antifÃngica de Calotropis procera." Universidade Federal do CearÃ, 2014. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=13438.
Full textAbstract:
Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgicoEm etapa anterior a este trabalho, uma proteÃna purificada a partir do lÃtex da planta Calotropis procera foi purificada e sua caracterizaÃÃo bioquÃmica revelou ser esta uma proteÃna similar a osmotinas e que a mesma, denominada de CpOsm, exibia forte atividade contra fungos fitopatogÃnicos. Neste trabalho foram investigados sistemas de expressÃo heterÃlogos, procarionte e eucarionte, com o objetivo de estabelecer sistemas de expressÃo que pudessem produzir CpOsm recombinante e avaliar se sua expressÃo produzia a proteÃna ativa, com aÃÃo antifÃngica. A partir do sequenciamento do cDNA da CpOsm e ensaios de cristalizaÃÃo desenvolvidos com a proteÃna purificada do lÃtex foi possÃvel estudar suas caracterÃsticas moleculares. Para a expressÃo em E. coli foi utilizado o vetor pET303CT-His e P. pastoris o vetor pPICZαA. A proteÃna recombinante (rCpOsm) expressa no sistema procarionte nÃo foi secretada para o meio externo, acumulando-se no espaÃo intracelular, formando corpos de inclusÃo, nos quais a proteÃna estava insolÃvel. Embora a insolubilidade represente um passo limitante, este sistema de expressÃo pode ser muito interessante para a produÃÃo quantitativa de rCpOsm para outros fins como produÃÃo de anticorpos ou estudos de folding/refolding proteico, considerado suas caracterÃsticas moleculares peculiares, observadas nos estudos cristalogrÃficos, tais como o conjunto de pontes dissulfeto intracadeia. rCpOsm foi tambÃm expressa em cÃlulas de P. pastoris, entretanto o sistema de expressÃo deverÃ, necessariamente, sofrer melhorias para maximizar o rendimento. rCpOsm de P. pastoris foi inicialmente detectada por sequenciamento de novo por espectrometria de massas a partir da digestÃo trÃptica. A identificaÃÃo de peptÃdeos internos confirmou sua presenÃa no meio extracelular. A limitaÃÃo deu-se pela baixa taxa de expressÃo, o que, por conseguinte, nÃo permitiu uma caracterizaÃÃo mais ampla da rCpOsm deste sistema. rCpOsm expressa em P. postoris estava em sua forma ativa. Em uma anÃlise comparativa, rCpOsm e CpOsm, de modo e intensidade similares, foram capazes de alterar drasticamente a morfologia de esporos de F. solani e reduzir seu volume, comparados a esporos nÃo tratados, revelado atravÃs de microscopia de forÃa atÃmica. CpOsm foi cristalizada pelo mÃtodo de gota pendente e os cristais obtidos difrataram a uma resoluÃÃo de 1,61 à com caracterÃsticas morfolÃgicas do espaÃo cristalogrÃfico P6122. Os dados coletados sugerem que a proteÃna mantÃm uma estrutura em monÃmero, correspondendo a sequÃncia de aminoÃcidos do cDNA, com 203 resÃduos. A estrutura pode ser vista como trÃs regiÃes distintas, presentes em outras osmotinas jà descritas, formando um domÃnio central onde hà um conjunto de folhas beta, sendo este o mais longo, e dois outros menores, formados de estrutura predominantemente desordenadas com pequenos segmentos em alfa-hÃlice (domÃnio II) e longas alÃas (domÃnio III). Oito pontes dissulfeto estabilizam a estrutura e envolvem todos os resÃduos de cisteÃna da estrutura primÃria. NÃo hà evidencias cristalogrÃficas para formaÃÃo de oligÃmeros. Este estudo conclui que a expressÃo heterÃloga em sistema eucarionte produz rCpOsm ativa e isto representa uma etapa a mais cumprida para a sua possÃvel expressÃo em plantas com o objetivo de proteÃÃo contra fitopatÃgenos.
In a previous step to this work, a latex protein belonging to Calotropis procera was described. The protein, named CpOsm, exhibited biochemical characteristics closely related to pathogenesis related proteins joined into PR-5 group. The new protein with osmotin characteristics displayed activity against phytopatogenic fungi. Here, attempts to obtain a suitable heterologous system to express functional recombinant CpOsm were performed in Prokaryote and Eukaryote expression systems. Further, cDNA sequencing and crystallographic assays were performed using CpOsm and the molecular and structural properties of the functional protein. The vector pET303CT-His and PICZαA were used to express CpOsm in E. coli and P. pastoris, respectively. The ecombinant protein (rCpOsm) produced in the Prokaryote system was retained into E. oli cells and deposited as inclusion bodies. rCpOsm was insoluble. Although insoluble roteins into inclusion bodies represent an adverse phase for obtaining the active CpOsm, this system of expression can be interesting to other goals as quantitative roduction of rCpOsm for producing antibodies or to study protein folding/refolding, ince CpOsm possesses peculiar structural characteristics such as occurrence of an xtended network or intra chain disulfide bonds stabilizing the overall structure. rCpOsm as also successfully expressed in P. pastoris. However, this protocol must to undergo improvement in order to maximize yield. rCpOsm was initially detected in the P. pastoris expression system by MS/MS de novo sequencing after tryptic digestion. Identification of internal peptides present in the extracellular media confirmed the production and excretion of rCpOsm in P. pastoris cells. The very low yield of recombinant protein avoided enough amount of purified rCpOsm to perform a broad characterization. On a comparative basis, native CpOsm, purified of the latex, and rCpOsm, purified from P. pastoris cultures, at similar mode and intensity were capable of drastically alter the morphological architecture of spores of F.solani and reduced their olume as compared to non-treated spores, as revealed by atomic force microscopy measurements. CpOsm was crystallized by the pendant drop method and the crystals grown diffracted at 1.61 Ã. They fit on the P6122 space. The data collecting, supported by the cDNA deduced amino acid sequence suggested that CpOsm occurs as an monomeric structure composed of a unique chain of 203 amino acid residues and no evidences for quaternary association was seen. The overall structure can separated in three structural domains, which have been reported in other osmotins. The central region preserves the set of beta-sheets and is the largest. The others exhibit short segments on alpha-helix interconnected by randomized sequences (domain II). In domain III predominates randomized sequences and long loops. Eight disulfide bonds stabilize the structure and involve all cysteine residues of the primary sequence. The heterologous expression of CpOsm on Eukaryote system produces rCpOsm active and this support the hypothesis that rCpOsm is a suitable candidate for heterologous expression in plants in order to obtain improved crops against selected phytopatogens.
2
Nepomuceno, Denise Rocha. "Clonagem e expressão de uma lectina de Artocarpus incisa L. (Frutalina) em Escherichia coli." reponame:Repositório Institucional da UFC, 2008. http://www.repositorio.ufc.br/handle/riufc/10819.
Full textAbstract:
NEPOMUCENO, D. R. Clonagem e expressão de uma lectina de Artocarpus incisa L. (Frutalina) em Escherichia coli. 2008. 81 f. Dissertação (Mestrado em Bioquímica) - Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2008.Submitted by Francisco Lacerda (lacerda@ufc.br) on 2014-11-27T19:23:51Z No. of bitstreams: 1 2008_dis_drnepomuceno.pdf: 815376 bytes, checksum: 17de51e5080e91f90b2ad84ac669dbba (MD5)
Approved for entry into archive by José Jairo Viana de Sousa(jairo@ufc.br) on 2015-02-27T19:42:31Z (GMT) No. of bitstreams: 1 2008_dis_drnepomuceno.pdf: 815376 bytes, checksum: 17de51e5080e91f90b2ad84ac669dbba (MD5)
Made available in DSpace on 2015-02-27T19:42:31Z (GMT). No. of bitstreams: 1 2008_dis_drnepomuceno.pdf: 815376 bytes, checksum: 17de51e5080e91f90b2ad84ac669dbba (MD5) Previous issue date: 2008
The lectins constitute biotechnological tools of fundamental importance in hystochemical and cytochemical studies for the detection of tissue glycoconjugates. Among their applications are the identification of membrane receptors and the detection of neoplastic characteristic structures. Frutalin, an -D-galactose-binding lectin from Artocarpus incise seeds, has already been used in the hystochemical detection of thyreoid and breast neoplasia. In this work was made a study of the cloning and expression of the frutalin gene in Escherichia coli Origami (DE3) cells and was determined the three-dimensional structure of this protein through homology modeling. The frutalin gene cloning was accomplished from the product on RT-PCR with seeds in distinct stages of development of the Artocarpus incisa fruits. Twelve clones were obtained, from which five were sequenced. The frutalin gene was expressed in E. coli using the pET15b expression vector. A recombinant lectin (rFrutalin) was expressed by growing the bacteria in the presence of isopropyl -D-thiogalactopyranoside 1mM. All the recombinant lectin was found in an insoluble aggregated form as inclusion bodies. The recombinant lectin had a higher molecular mass (19,2 kDa) than the native lectin (15 kDa) as estimated by SDS-PAGE and Western blot analyses, showing that the recombinant single chain Frutalin is not processed in E. coli cells. The models generated for the clones obtained indicate that the mutations do not change the molecules active sites. Mutations in the clones obtained suggest the occurrence of isoforms of this protein.
As lectinas constituem ferramentas biotecnológicas de fundamental importância em estudos histoquímicos e citoquímicos para a detecção de glicoconjugados nos tecidos. Entre estas aplicações, destacam-se a identificação de receptores de membrana e a detecção de estruturas características de neoplasias. A Frutalina, uma lectina -D-galactose-ligante encontrada em sementes de Artocarpus incisa, já foi utilizada na detecção histoquímica de lesões malignas de mama e tireóide. No presente trabalho foi realizada a clonagem e expressão do gene da frutalina em células de Escherichia coli Origami (DE3) e foi determinada a estrutura tridimensional dessa proteína através da modelagem por homologia. A clonagem do gene da frutalina foi realizada a partir do produto amplificado da RT-PCR de sementes em diferentes estágios de maturação dos frutos de A. incisa. Foram obtidos 12 clones, dos quais 5 foram seqüenciados. A lectina recombinante foi expressa como cadeia única, formada pela cadeia beta com 20 resíduos, ligada a um peptídeo de ligação com quatro resíduos, e a cadeia alfa com 133 resíduos. A expressão da lectina foi indicada pelo aparecimento de uma banda protéica com massa molecular aparente de 19,2 kDa, superior a da lectina nativa (15 kDa), após a indução com IPTG 1mM. A lectina recombinante foi mantida exclusivamente em corpos de inclusão e foi reconhecida imunologicamente por anticorpos policlonais anti-Frutalina. Os modelos gerados para os clones obtidos indicam que as mutações ocorridas não alteram os sítios de ligação a carboidratos das moléculas. As mutações nos clones obtidos sugerem a ocorrência de isoformas dessa proteína.
3
Landim, PatrÃcia Gadelha de Castro. "ProduÃÃo em Pichia pastoris de uma quitinase de feijÃo-de-corda com atividade antifÃngica." Universidade Federal do CearÃ, 2011. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=8584.
Full textAbstract:
Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgicoAs quitinases sÃo enzimas capazes de hidrolisar as ligaÃÃes β-(1,4)-glicosÃdicas presentes em biopolÃmeros de N-acetil-β-D-glucosamina, principalmente quitina, um polissacarÃdeo estrutural presente na parede celular de diversos fungos. No presente trabalho, uma quitinase de classe I de feijÃo-de-corda (Vigna unguiculata) foi expressa em sistemas heterÃlogos e a proteÃna recombinante (rVuChi) foi caracterizada bioquimicamente bem como em relaÃÃo ao seu efeito sobre o crescimento micelial e germinaÃÃo de esporos/conÃdios de fungos filamentosos. A seqÃÃncia de DNA codificando a proteÃna foi amplificada por PCR e clonada nos vetores de expressÃo pET32a(+) e pPICZαA, para expressÃo heterÃloga em Escherichia coli e Pichia pastoris, respectivamente. A expressÃo de rVuChi em cÃlulas de E. coli ArticExpress DE3 se deu em corpos de inclusÃo. Em seis estirpes de P. pastoris a proteÃna recombinante foi secretada, de forma solÃvel, para o meio de cultura. Na fraÃÃo extracelular da estirpe KM71H foi observada a maior atividade quitinolÃtica, apÃs 72 horas de induÃÃo. A detecÃÃo de rVuChi foi feita por SDS-PAGE e com o kit Invision His-Tag stain, onde foram identificadas duas bandas protÃicas de massas moleculares aparentes de 30 e 33 kDa. Ambas as bandas apresentaram a mesma sequÃncia N-terminal e a ausÃncia de N-glicosilaÃÃo foi verificada. A quitinase recombinante estava presente principalmente na fraÃÃo F0/40 precipitada com sulfato de amÃnio e foi purificada a homogeneidade tanto por cromatografia de afinidade em matriz de quitina (com rendimento de 18,31 mg por litro de meio de cultura), quanto por cromatografia de interaÃÃes hidrofÃbicas em coluna de Phenyl Sepharose CL-4B (rendimento de 13,2 mg/L), seguidas de ultrafiltraÃÃo em membrana com limite de exclusÃo de 50 kDa. A rVuChi apresentou atividades endo e exo-quitinolÃtica frente a quitina coloidal e hidrolisou glicol-quitina em gel de SDS-PAGE, embora nÃo tenha apresentado atividade contra substratos sintÃticos contendo p-nitrofenol. A quitinase purificada apresentou massa molecular de 32 e 33,1 kDa por cromatografia de exclusÃo molecular em colunas de Superose 12 HR e Superdex 200, respectivamente. Em gel bidimensional, rVuChi apresentou um conjunto de seis âspotsâ com pI entre 4,44 e 5,15. A quitinase mostrou-se ainda termoestÃvel em temperaturas atà 50 ÂC e sua atividade enzimÃtica mÃxima ocorreu em pH 5. Em geral, a presenÃa de Ãons metÃlicos causou uma reduÃÃo de sua atividade enzimÃtica. O agente quelante EDTA (0,5%) estimulou a atividade enzimÃtica enquanto que o detergente SDS (0,5%) a inibiu totalmente. A quitinase recombinante apresentou 37% de hÃlice alfa e 26% de folha beta, como determinado por espectroscopia de dicroÃsmo circular. A desnaturaÃÃo de 50% das molÃculas de rVuChi ocorreu a 54,41 ÂC. Os espectros de fluorescÃncia revelaram que a proteÃna produzida em P. pastoris estava em sua conformaÃÃo totalmente enovelada. A quitinase recombinante de feijÃo-de-corda foi capaz de inibir totalmente a germinaÃÃo de esporos de Penicillium herquei atà 48 horas, na dose de 100 μg e causou inibiÃÃo de 68%, nas doses de 50, 25 e 12,5 μg. Na dose de 150 μg, houve uma inibiÃÃo de 55% na germinaÃÃo dos conÃdios de Rhizoctonia solani e um leve efeito sobre a germinaÃÃo dos esporos de Colletotrichum lindemuthianum e C. musae. Nenhum efeito da rVuChi foi observado sobre a germinaÃÃo de esporos dos fungos C. gloeosporioides, Fusarium solani e F. oxysporum. AlÃm disso, a proteÃna recombinante retardou o crescimento micelial de P. herquei em aproximadamente 50% (100 μg), porÃm nÃo apresentou efeito sobre o crescimento micelial dos demais fungos. Desta forma, a quitinase classe I de V. unguiculata à uma proteÃna com atividade antifÃngica.
Chitinases are enzymes that hydrolyze the β-(1,4) glycosidic bonds present in biopolymers of N-acety-β-D-glucosamine, mainly chitin, a structural polysaccharide which is found in cell walls of several fungi. In plants, chitinases play a role as defense proteins against the attack of pests and pathogens. In this work, a class I chitinase from cowpea (Vigna unguiculata) was expressed in heterologous systems. The recombinant protein (rVuChi) was purified, and characterized biochemically and in relation to its effects on mycelial growth and germination of spores/conidia of filamentous fungi. The DNA coding sequence of the cowpea chitinase was amplified by PCR and the products cloned in the expression vectors pET32a(+) and pPICZαA, for heterologous expression in Escherichia coli and Pichia pastoris, respectively. In E. coli cells, the recombinant fusion protein occurred mainly as inclusion bodies. On the other hand, in six strains of P. pastoris, the recombinant cowpea chitinase was secreted in a soluble form into the culture medium. The highest chitinase activity was detected in the extracellular fraction of KM71H strain, 72 hours after induction. The recombinant VuChi was detected by SDS-PAGE and Invision His-Tag stain kit, which identified two protein bands with apparent molecular masses of 30 and 33 kDa. These two protein bands showed the same N-terminal sequence, and an absence of N-glycosylation. Most recombinant chitinase secreted into the culture medium was recovered in the fraction F0/40, precipitated with ammonium sulfate. The expressed protein was purified to homogeneity by affinity chromatography on chitin matrix (yield of 18.31 mg per liter of culture medium), or by hydrophobic interactions chromatography on a column of Phenyl Sepharose CL-4B (yield = 13.2 mg/L), followed by ultrafiltration in a membrane with exclusion limit of 50 kDa. The purified rVuChi was able to hydrolyze colloidal chitin (in solution) as well as glycol chitin (in SDS-PAGE), although it did not show enzymatic activity against synthetic substrates containing p-nitrophenol. The purified chitinase showed molecular masses of 32 and 33.1 kDa by size exclusion chromatography on columns of Superose 12 HR and Superdex 200, respectively. When submitted to 2D electrophoresis, rVuChi presented a set of six spots with pI values between 4.44 and 5.15. The chitinase was thermostable at temperatures up to 50 Â C and the enzyme activity was highest at pH 5. In general, the presence of metal ions caused a reduction of its enzymatic activity. The chelating agent EDTA (0.5%) stimulated the enzyme activity, whereas in the presence of the detergent SDS (0.5%) the rVuChi activity was completely inhibited. The recombinant chitinase showed 37% of alpha helix and 26% of beta sheet, as determined by circular dichroism spectroscopy. Denaturing of 50% of the rVuChi molecules occurs at 54.41 Â C. The fluorescence spectra showed that the protein produced in P. pastoris was in its fully folded conformation. The recombinant cowpea chitinase was able to completely inhibit the germination of spores of Penicillium herquei, after 48 hours, at a dose of 100 mg, and caused 68% inhibition at doses of 50, 25 and 12.5 mg. At a dose of 150 mg, there was 55% inhibition on conidial germination of Rhizoctonia solani and a slight effect on spore germination of Colletotrichum lindemuthianum and C. musae. There was no effect of rVuChi on spore germination of C. gloeosporioides, Fusarium solani and F. oxysporum. In addition, the recombinant protein delayed the mycelial growth of P. herquei in approximately 50% (at the dose of 100 mg) but had no effect on mycelial growth of the other fungi. Therefore, the cowpea class I chitinase is a protein with anti-fungal activity.
4
Viana, MartÃnio Ponte. "ToxicoproteÃmica aplicada à anÃlise de risco da proteÃna recombinante cry1ac de Bacillus thuringiensis." Universidade Federal do CearÃ, 2014. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=17262.
Full textAbstract:
CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel SuperiorCoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Dentre os vÃrios microrganismos entomopatogÃnicos utilizados no controle biolÃgico, o Bacillus thuringiensis (Bt) vem sendo considerado uma das alternativas mais viÃveis, devido à presenÃa de proteÃnas inseticidas em seus esporos, como as δ-endotoxinas Cry. As toxinas Cry apresentam atividade contra diferentes ordens de insetos, como Coleoptera, Diptera, Hymenoptera, Hemiptera e Lepidoptera. Tais proteÃnas sÃo altamente especÃficas, ou seja, sÃo inÃcuas para a maioria dos organismos nÃo-alvo, fato que favorece sua utilizaÃÃo na agricultura. Hoje existem mais de 114 milhÃes de hectares de lavouras geneticamente modificadas e 37% expressam traÃos de proteÃnas inseticidas de Bt. Como os organismos geneticamente modificados (OGMs) estÃo se tornando cada vez mais predominantes, vÃrias organizaÃÃes internacionais tÃm dado orientaÃÃes no sentido de investigar a seguranÃa de alimentos provenientes de OGMs. Este trabalho teve como objetivo utilizar uma abordagem toxicoproteÃmica para anÃlise de risco da proteÃna recombinante Cry1Ac de Bt a fim de contribuir para uma maior compreensÃo de seus efeitos em modelo de mamÃferos. O ensaio de toxicidade foi conduzido de acordo com o protocolo 425 da âOrganizaÃÃo para a CooperaÃÃo e Desenvolvimento EconÃmicoâ - OECD e nÃo foram verificadas mortes ou sinais de toxicidade. As anÃlises proteÃmicas baseadas em gel mostraram 4 proteÃnas diferencialmente expressas Serpina α-1, Serpina A3K, CininogÃnio e Complemento C3. Essas proteÃnas tiveram uma reduÃÃo em sua expressÃo no grupo tratado com a toxina Cry1Ac. Na abordagem âgel-freeâ foram identificadas 7 proteÃnas diferencialmente expressas. Dentre elas, fator I, inibidor de tripsina H3, plasminogÃnio, serpina A6, albumina e proteÃna resistente à oxidaÃÃo apresentaram uma expressÃo maior em animais tratados com Cry1Ac, enquanto que a protrombina teve sua expressÃo reduzida em animais tratados com a mesma proteÃna. Tais molÃculas sÃo importantes para hemostasia e sistema imune, podendo interferir no processo inflamatÃrio, na ativaÃÃo da via do complemento e da cascata de coagulaÃÃo. No entanto, a abordagem toxicoproteÃmica adotada mostra-se Ãtil para a identificaÃÃo de efeitos adversos, atà mesmo de uma jà amplamente conhecida por sua baixa toxicidade. Isso encoraja a utilizaÃÃo da referida abordagem para a avaliaÃÃo de risco de proteÃnas recombinantes. Apesar das alteraÃÃes fisiolÃgicas causadas, a proteÃna Cry1Ac ainda à considerada segura jà que tais alteraÃÃes ocorreram na dose mais alta recomenda pela OECD (2000mg/Kg) e sem causar morte dos animais.
Among the various pathogenic bacteria used in biological control, Bacillus thuringiensis has been considered one of the most viable alternatives, due to the presence of insecticidal proteins in their spores, such as the δ-endotoxin, Cry. The Cry toxins exhibit activity against different insect orders, such as Coleoptera, Diptera, Hymenoptera, Hemiptera and Lepidoptera. Such proteins are highly specific, which means, they are harmless to most organisms. This fact justifies their use in agriculture. Nowadays, there are over 114 million hectares of GM crops and 37% express traits of insecticidal proteins of B. thuringiensis. Since genetically modified organisms (GMOs) are becoming increasingly prevalent, several international organizations have given guidelines to investigate the safety of food derived from GMOs. This work aimed to evaluate the acute toxicity of the entomotoxin Cry1Ac in rats through classical in vivo analyzes associated with proteomic study by two-dimensional electrophoresis and shotgum proteomic technique (gel-free). Toxicity tests were conducted according to the protocol of 425 "Organization of Economic Cooperation and Development" - OECD and no deaths or signs of toxicity were observed. The gel-based proteomic analysis showed four differentially expressed proteins: Serpin α-1, Serpin A3K, Kininogen and Complement C3. These proteins expressions were reduced for the group treated with the Cry1Ac toxin. In gel-free approach, seven differentially expressed proteins were identified. Among them, factor I, H3 trypsin inhibitor, plasminogen, serpin A6, albumin, and protein resistant to oxidation showed a higher expression in animals treated with Cry1Ac, while the prothrombin expression was reduced in animals treated with the same protein. Such molecules are important for hemostasis and immune system, and may interfere with the inflammatory process, activation of the complement pathway and the coagulation cascade. Despite the physiological changes caused by Cry1Ac, this protein is still considered safe since these changes occurred at the highest dose recommended by OECD (2000 mg / kg) and without causing death of the animals.
5
Teixeira, CÃcero Silvano. "ExpressÃo de uma quitinase de Chromobacterium Violaceum em Pichia Pastoris: purificaÃÃo e caracterizaÃÃo parcial da proteÃna recombinante." Universidade Federal do CearÃ, 2011. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=5880.
Full textAbstract:
FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgicoA utilizaÃÃo de microrganismos como sistemas heterÃlogos de expressÃo de proteÃnas tem se mostrado uma estratÃgia alternativa e/ou complementar aos passos tradicionais utilizados no processo de purificaÃÃo de proteÃnas. Diante deste contexto, quitinases (EC 3.2.1.14), enzimas hidrolÃticas capazes de degradar quitina, tÃm sido expressas em diferentes sistemas heterÃlogos, incluindo bactÃrias e leveduras. Quitina à um polÃmero linear composto de resÃduos de N-acetil-β-D-glucosamina (GlcNAc), sendo um importante constituinte estrutural da carapaÃa de crustÃceos e membrana peritrÃfica de insetos e, ainda, da parede celular de fungos. Este trabalho teve por objetivo expressar uma quitinase, codificada pela ORF cv3316, de Chromobacterium violaceum ATCC 12472 na levedura metilotrÃfica Pichia pastoris, estirpes GS115 e KM71H, alÃm de purificar e caracterizar a proteÃna recombinante (rCHI3316). A estirpe GS115, portando a construÃÃo (pPICZαA-CV3316), foi selecionada para os experimentos posteriores, pois apresentou um maior nÃvel de expressÃo quando comparada à cepa KM71H. Cromatografia de afinidade em coluna de nÃquel imobilizado foi utilizada para purificar a quitinase recombinante (rCHI3316), que foi eluÃda como um Ãnico pico com imidazol 0,04 M. A proteÃna purificada se mostrou homogÃnea quando submetida à eletroforese em gel de poliacrilamida 15% em presenÃa de SDS e -mercaptoetanol (SDS-PAGE). Nessas condiÃÃes, uma Ãnica banda com massa molecular aparente de aproximadamente 87 kDa foi observada. A quitinase rCHI3316 de C. violaceum foi expressa de forma solÃvel utilizando o sistema de expressÃo P. pastoris e, alÃm disso, sua purificaÃÃo foi realizada de forma satisfatÃria. O conteÃdo de estrutura secundÃria foi estimado por espectroscopia de dicroÃsmo circular (CD), com a proteÃna submetida a diferentes temperaturas (10-90 ÂC). Na temperatura de 24 ÂC, o espectro de CD revelou a predominÃncia do conteÃdo de hÃlice alfa (38%), folha beta (26 %) e estrutura randÃmica (37%). Entre as temperaturas de 10-50 ÂC, rCHI3316 exibiu um espectro caracterÃstico de folha beta. A partir de 60 ÂC atà 90 ÂC, rCHI3316 adquiriu uma conformaÃÃo caracterÃstica de hÃlice alfa. A temperatura mÃdia para essa transiÃÃo conformacional foi calculada como sendo 59,6 1,21 ÂC. Experimentos de espectroscopia de fluorescÃncia, com excitaÃÃo a 280 e 290 nm, produziram espectros de emissÃo com comprimentos de onda mÃximos iguais a 339 e 342 nm, respectivamente. Esses valores sÃo caracterÃsticos de resÃduos de triptofano parcialmente expostos ao solvente. Atividade quitinolÃtica contra vÃrios substratos e dependÃncia de pH da atividade enzimÃtica da proteÃna pura foram avaliados. A rCHI3316 exibiu atividade hidrolÃtica sobre os substratos quitina coloidal (1.189,40 U/mgP), 4-nitrofenil N-Nâ-diacetil-β-D-quitobiosÃdeo (30.411,0 U/mgP) e 4-nitrofenil N-Nâ-Nââ-triacetilquitotriose (13.150,0 U/mgP); entretanto, nenhuma atividade enzimÃtica da rCHI3316 foi detectada frente a 4-nitofenil N-acetil-β-D-glucosaminÃdeo. A enzima exibiu atividade quitinolÃtica Ãtima (100%) em pH 5,0; quando quitina coloidal foi utilizada como substrato. Em adiÃÃo, a atividade antifÃngica contra fungos fitopatogÃnicos, Fusarium solani, Fusarium oxysporum, Rhizoctonia solani e Penicillium herquei foi investigada. rCHI3316 nÃo inibiu a germinaÃÃo e o crescimento micelial dos esporos dos fungos testados, na concentraÃÃo de 0,63 mgP.ml-1. Estudos subseqÃentes deverÃo ser realizados na intenÃÃo de descobrir potenciais aplicaÃÃes desta proteÃna como uma ferramenta biolÃgica no controle de outros fungos fitopatogÃnicos bem como insetos considerados pragas.
Microorganisms are a valuable tool for the expression of proteins from a variety of sources, including plants, animals and other microorganisms. Thus, chitinases, a group of glycosil hydrolases capable to hydrolize chitin, have already been expressed and purified from different systems including bacteria and yeast. Chitin, a linear polymer of N-acetyl-β-D-glucosamine (GlcNAc), is an important structural component found in the crustacean shells, in the peritrophic membrane of insect guts as well as in the fungi cell walls. The aim of this work was to express a chitinase (encoded by the ORF CV3316) from Chromobacterium violaceum ATCC 12472, using the methylotrophic yeast Pichia pastoris strains GS115 and KM71H. Furthermore, purification and partial characterization of the recombinant protein were also achieved. The GS115 strain carrying the expression cassette pPICZαA-CV3316 was selected due to its higher expression level as compared to KM71H strain. Immobilized metal ion affinity chromatography was employed to purify the recombinant chitinase which was eluted as a single peak at 0.04 M imidazol. The homogeneity of the purified protein was confirmed as judged by polyacrylamide gel electrophoresis (SDS-PAGE). In these conditions, the recombinant chitinase migrated as a single protein band with an apparent molecular mass of about 87 kDa. Thus, a chitinase from C. violaceum ATCC 12472 was successfully expressed in P. pastoris and the soluble recombinant protein purified. The content of secondary structure was investigated by circular dichroism (CD) spectroscopy. At 24 oC the CD spectrum revealed secondary structure contents of 37% (alpha helix), 26% (beta sheet) and 38% (random coil). The CD spectra obtained in the temperature range 10-50 oC were characteristic of beta sheet. In contrast, the CD spectra generated in the range 60-90 oC were characteristic of alpha helix. The midpoint temperature of this conformational transition was 59.6 1.2 oC as calculated from the CD experimental data. Fluorescence spectroscopy was carried out with excitation at 280 and 290 nm, producing emission spectra in which the wavelengths of maximum emission were 339 and 342 nm, respectively. This behavior is characteristic of tryptophan residues in limited contact with water. Chitinolytic activity against several substrates and the pH dependency of the enzymatic activity of the pure protein were all accessed. The purified enzyme showed hydrolytic activity on the following substrates: colloidal chitin (1,189.4 U.mgP-1), 4-nitrophenyl N-Nâ-diacetyl--D-chitobioside (30,411.0 U.mgP-1) and 4-nitrophenyl β-D-N-Nâ-Nâ-triacetylchitotriose (13,150.0 U.mgP-1); and, respectively. In contrast, no activity was detected using 4-nitrophenyl N-acetyl-β-D-glucosaminide as substrate. The enzyme presented an optimal chitinolytic activity at pH 5.0 using colloidal chitin as a substrate. Additionally, the antifungal activity against the phytopathogenic fungi, Fusarium solani, Fusarium oxysporum, Rhizoctonia solani and Penicillium herquei was investigated. The recombinant chitinase did not inhibit the spore germination and the mycelium growth of the tested fungi, at the 0.63 mgP.ml-1 concentration. Further studies should be carried out in order to discover potential applications of this protein as a biotechnological tool in the control of other phytopathogenic fungi as well as economically important pests.
6
Koscky, Paier Carlos Roberto 1983. "Padronização da expressão heterologa e de modelo de ensaio de atividade para a proteina quinase humana S6K." [s.n.], 2009. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314787.
Full textAbstract:
Orientador: Nilson Ivo Tonin ZanchinDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-14T12:40:52Z (GMT). No. of bitstreams: 1 KosckyPaier_CarlosRoberto_M.pdf: 3760581 bytes, checksum: 99331529324819b59a4360d60efd9b9a (MD5) Previous issue date: 2009
Resumo: A quinase de 70 kDa da proteína ribossomal S6, isoforma 1 (S6K1), é uma fosfoproteína implicada na regulação de genes relacionados ao controle da tradução em mamíferos e possui uma forma nuclear (a1) e uma citoplasmática (a2). A fosforilação do seu principal alvo, a proteína RPS6, tem sido comumente associada ao recrutamento seletivo dos 5'-TOP (5' tract of oligopyrimidine) mRNAs pela maquinaria de tradução, embora haja estudos contrariando esta hipótese. Devido às funções de seus demais alvos, S6K1 tem sido implicada na sobrevivência celular e em diversos outros processos, como crescimento, câncer e resistência à insulina. S6K1 é ativada por um mecanismo que envolve fosforilação seqüencial através da ativação das vias mTORC1 (complexo 1 do alvo da rapamicina em mamíferos) e PI3K (fosfoinositol-3 quinase). Como uma quinase da família AGC, S6K1 deve ser fosforilada por mTORC1 no resíduo Thr389 do domínio hidrofóbico e, em seguida, por PDPK1 (proteína quinase 1 dependente de fosfoinositol) no resíduo Thr229 da alça T do domínio catalítico. Estes eventos ocorrem somente após a fosforilação em diversos sítios do domínio auto-inibitório carboxiterminal, por mTORC1. O objetivo deste trabalho foi desenvolver um ensaio modelo para análise da função da S6K1 in vitro e utilizá-lo como ferramenta na elucidação do papel de proteínas adaptadoras da via de mTOR em interações com a S6K1. Para isso foi necessário produzir as proteínas recombinantes para ensaios de interação e para realização de um ensaio de atividade para a S6K1. Foram testados vários sistemas de expressão para Escherichia coli para produção das construções GST-S6K1a1-His6, GST-S6K1a2-His6 e GST-S6K1a2T389E?CT (forma a2 de S6K1 com a substituição T389E e o carboxiterminal truncado), GST-PDPK1 e GST-CDPDPK1 (domínio catalítico de PDPK1 fusionado a GST). A expressão das formas truncadas de S6K1 e PDPK1 foi mais eficiente em E. coli. Embora o rendimento tenha ficado muito aquém do esperado, foi suficiente para os ensaios de interação in vitro. Também foi feita a expressão em E. coli da região C-terminal da proteína RPS6, que é o substrato da S6K1, em fusão com a proteína D do fago ?. Posteriormente, foram montados sistemas de expressão das construções His6-S6K1a2T389E?CT e His6-CDPDPK1 em células de inseto, a partir de vetor de baculovírus. Constatou-se que essas construções são expressas na forma de fosfoproteínas em células de inseto. Ensaios de GST pull-down com GST-S6K1a2-His6 e GST-S6K1a2T389E?CT contra as duas isoformas da subunidade catalítica da PP2AC, His6-PP2ACa(maior) e His6-PP2ACa(menor), revelaram que His6-PP2ACa(maior) não interage com GST-S6K1a2-His6, embora interaja fortemente com GST-S6K1a2T389E?CT. Já a construção His6-PP2ACa(menor) interage fracamente com as construções GST-S6K1a2-His6 e GST-S6K1a2T389E?CT. Tomados em conjunto, os resultados sugerem que a presença do C-terminal não fosforilado de S6K1a2 impede a interação com PP2ACa(maior). PP2ACa(menor) comporta-se de forma completamente diferente da isoforma maior, pois a interação entre PP2ACa(menor) e S6K1a2 parece ser independente do carboxiterminal da quinase, visto que as quantidades de S6K1a2T389E?CT e de S6K1a2 inteira que interagem com PP2ACa(menor) são semelhantes. Esses resultados necessitam ainda serem confirmados in vivo. Outros experimentos de GST pull-down confirmaram que as construções de S6K1 não interagem com a4, embora interajam com TIPRL1. Se confirmado in vivo, esse resultado compõe um novo quadro na regulação coordenada entre mTOR1 e PP2A, do qual TIPRL1 parece participar. As construções genéticas e os sistemas de expressão gerados neste trabalho possibilitaram a obtenção dos reagentes necessários para analisar o mecanismo de regulação da quinase S6K1, mediado por proteínas regulatórias. Permitem também desenvolver uma série de experimentos, como busca de inibidores específicos para a S6K1, que dependem da reconstituição de ensaios de atividade in vitro com a S6K1 ativada. Contudo, o ensaio de atividade realizado não apresentou resultados satisfatórios e precisa ser desenvolvido.
Abstract: The 70kDa ribosomal S6 protein kinase 1 (S6K1) is a phosphoprotein involved in the regulation of genes related to translational control in mammals. S6K1 shows distinct nuclear (a1) and cytoplasmic (a2) forms. Phosphorylation of the S6K1 best characterized target, the protein of the small ribosomal subunit (RPS6), has been generally associated to the selective recruitment of the 5'-TOP mRNAs (5' tract of oligopyrimidine) by the translational machinery, although there is still some controversy on this issue. Due to the function of its targets, S6K1 has been implicated in several cellular processes including cell growth, cancer and insulin resistance. S6K1 is activated by a mechanism of sequential phosphorylation following activation of the mTORC1 (mammalian target of rapamycin complex 1) and PI3K (phosphoinositide-3-kinase) pathways. As a kinase of the AGC family, S6K1 activation requires mTORC1 phosphorylation of residue Thr389 of the hydrophobic domain followed by PDPK1 (phosphoinositide dependent protein kinase 1) phosphorylation of residue Thr229 at the T loop of the catalytic domain. These take place only after phosphorylation by mTORC1 of several residues of the autoinhibitory C-terminal domain. The objective of this work was to develop an assay to analyze the function of S6K1 in vitro and use it as a tool in the discovering of the functions of regulators proteins of the mTOR cascade in interactions with S6K1. For these purposes, expression systems were constructed to produce the various recombinant proteins to be used in the interaction and activity assays. Several genetic constructions were tested in Escherichia coli for the production of GST-S6K1a1-His6, GST-S6K1a2-His6 and GST-S6K1a2T389E?CT (a2 form of S6K1 with the T389E substitution and truncated carboxiterminus), GST-PDPK1 and GST-CDPDPK1 (GST fusion protein of the catalytic domain of PDPK1). The truncated forms were expressed more efficiently in E. coli. Although the yield in E. coli was lower than expected, it was sufficient to perform interaction assays. The C-terminal domain of RPS6, a substrate for S6K1, was successfully expressed in E. coli as a fusion protein with the phage ? protein D. Subsequently, expression systems for production of His6-S6K1a2T389E?CT and His6-CDPDPK1 in insect cells were constructed using baculovirus vectors. It was found that these constructs are expressed in the form of phosphoproteins in insect cells. GST pull-down assays using GST-S6K1a2-His6 e GST-S6K1a2T389E?CT to test interaction with the PP2AC isoforms His6-PP2ACa(major) and His6-PP2ACa(minor) revealed that His6-PP2ACa(major) does not interact with GST-S6K1a2-His6, although it interacts strongly with GST-S6K1a2T389E?CT. On the other hand, His6-PP2ACa(minor) interacts weakly with both GST- S6K1a2-His6 and GST-S6K1a2T389E?CT. This finding suggests that the unphosphorylated C-terminal of S6K1a2 inhibits interaction with PP2ACa(major). His6-PP2ACa(minor) behaves differently form His6-PP2ACa(major). Its interaction with S6K1a2 seems to be independent of the C-terminal since the amounts of S6K1a2T389E?CT and S6K1a2 that interact with His6-PP2ACa(minor) are similar. Future work in vivo is required to confirm these results. GST pull-down assays confirmed that a4 does not interact with the constructions of S6K1, while TIPRL1 interacts with them. If confirmed in vivo, these results provides a new perspective for the coordinated regulation between mTOR1 and PP2A, which apparently involves also TIPRL1. The genetic constructions and expression systems established in this work allow the production of the reagents required to study the mechanism of S6K1 regulation mediated by adaptor proteins. They will also allow the development of experiments such as screening for specific S6K1 inhibitors, which depend on reconstitution of S6K1 activity assays using activated S6K1. Nevertheless, the activity assay performed did not yield satisfactory outcomes and must be improved.
Mestrado
Bioquimica
Mestre em Biologia Funcional e Molecular
7
Roca, Pinilla Ramon. "Development of a new generation of antimicrobial proteins based on a versatile nanoparticulated format and multidomain structure." Doctoral thesis, Universitat Autònoma de Barcelona, 2020. http://hdl.handle.net/10803/670790.
Full textAbstract:
Durant la major part de la historia humana, els patògens han estat una de les principals causes de morts i malalties. Gràcies al descobriment dels antibiòtics hem aconseguit tractar aquestes malalties amb facilitat, però el seu mal ús ha accelerat l’aparició de resistències als antimicrobians (AMRs). Atès que les AMRs han provocat que la majoria de fàrmacs antimicrobians siguin ineficaços, el desenvolupament de tractaments alternatius és mes necessari que mai. Els pèptids de la defensa del hoste (HDPs) han estat proposats com a models per la generació de nous antimicrobians per lluitar contra les infeccions AMR. Tot i així, la majoria d’HDPs és produeixen mitjançant la síntesi química, un procés que és car, insostenible i difícil d’escalar. Alternativament, la producció recombinant d’HDPs és molt atractiva però complicada, ja que són pèptids altament susceptibles de ser degradats i són tòxics per l’hoste recombinant. Malgrat això, els cossos d’inclusió (IBs), que són agregats de proteïna formats durant els processos de producció recombinant, es poden utilitzar com a format alternatiu al de la proteïna soluble per permetre la producció d’HDPs dins l’hoste sense efectes tòxics. D’altra banda, la construcció de proteïnes quimèriques podria ser una estratègia per expressar pèptids petits amb èxit. En aquest context, aquesta tesi explora diverses estratègies per la producció recombinant d’HDPs. Per una banda, hem explorat l’ús de les cremalleres de leucina com a dominis potencials per fomentar la producció recombinant d’HDPs en la fracció insoluble i per augmentar la qualitat de la proteïna recombinant dels IBs. A més a més, hem desenvolupat diverses proteïnes antimicrobianes multidomini basades en la fusió de diferents pèptids HDP i proteïnes de la immunitat innata. Com que també hem utilitzat cremalleres de leucina en aquests constructes, es poden expressar de manera efectiva – sense toxicitat per la cèl·lula productora. A més, en cas de necessitat, podem recuperar antimicrobians solubles a partir dels IBs gràcies a un protocol de solubilització suau i no desnaturalitzant. En conjunt, hem demostrat que aquests constructes tenen un ampli espectre d’acció antimicrobiana contra bacteris multi resistents (MDR), tant en el format soluble com en el format d´IB. És més, els constructes també són capaços d’estimular l’alliberament de IL-8 dins d’un potencial rang de propietats immunomoduladores. Aquests resultats ens han convidat a utilitzar les nostres proteïnes en la biofuncionalització de monocapes autoacoblants per evitar la formació de biofilms, i hem observat que aquestes proteïnes poden ancorar-se a aquests materials i evitar el creixement de biofilms. En resum, aquests resultats reforcen les proteïnes antimicrobianes multidomini com a potencials alternatives antimicrobianes amb propietat immunomoduladores.Durante la mayor parte de la historia humana, los patógenos han sido una de las principales causas de muertes y enfermedades. Gracias al descubrimiento de los antibióticos hemos conseguido tratar estas enfermedades con facilidad, pero su mal uso ha acelerado la aparición de resistencias a los antimicrobianos (AMRs). Dado que las AMRs han provocado que la mayoría de fármacos antimicrobianos sean ineficaces, el desarrollo de tratamientos alternativos es más necesario que nunca. Los péptidos de la defensa del huésped (HDPs) han sido propuestos como modelos para la generación de nuevos antimicrobianos para luchar contra las infecciones AMR. Sin embargo, la mayoría de HDPs se producen mediante la síntesis química, un proceso que es caro, insostenible y difícil de escalar. Alternativamente, la producción recombinante de HDPs es muy atractiva pero complicada, ya que son péptidos altamente susceptibles de ser degradados y son tóxicos para el huésped recombinante. Sin embargo, los cuerpos de inclusión (IBs), que son agregados de proteína formados durante los procesos de producción recombinante, se pueden utilizar como formato alternativo al de la proteína soluble para permitir la producción de HDPs dentro del huésped sin efectos tóxicos. Por otra parte, la construcción de proteínas quiméricas podría ser una estrategia para expresar péptidos pequeños con éxito. En este contexto, esta tesis explora diversas estrategias para la producción recombinante de HDPs. Por un lado, hemos explorado el uso de las cremalleras de leucina como dominios potenciales para fomentar la producción recombinante de HDPs en la fracción insoluble y para aumentar la calidad de la proteína recombinante en los IBs. Además, hemos desarrollado varias proteínas antimicrobianas multidominio basadas en la fusión de diferentes péptidos HDP y proteínas de la inmunidad innata. Como también hemos utilizado cremalleras de leucina en estos constructos, se pueden expresar de manera efectiva - sin toxicidad para la célula productora. Además, en caso de necesidad, podemos recuperar antimicrobianos solubles a partir de los IBs gracias a un protocolo de solubilización suave y no desnaturalizando. En conjunto, hemos demostrado que estos constructos tienen un amplio espectro de acción antimicrobiana contra bacterias multi resistentes (MDR), tanto en el formato soluble como en el formato de IBs. Es más, los constructos también son capaces de estimular la liberación de IL-8 dentro de un potencial rango de propiedades inmunomoduladoras. Estos resultados nos han invitado a utilizar nuestras proteínas en la biofuncionalizacón de monocapas autoensamblantes para evitar la formación de biofilms, y hemos observado que estas proteínas pueden anclarse a estos materiales y evitar el crecimiento de biofilms. En resumen, estos resultados refuerzan las proteínas antimicrobianas multidominio como potenciales alternativas antimicrobianas con propiedades inmunomoduladoras.
For most of human history, pathogens have been a leading cause of death and illness. Although we have attained the ability to treat them easily, thanks to the discovery of antibiotics, the widespread overuse and misuse of antimicrobial drugs have accelerated the appearance of antimicrobial resistances (AMRs). Because AMRs have rendered most antimicrobial drugs ineffective, the development of alternative approaches is more necessary than ever before. Host defense peptides (HDPs) have been proposed as blueprints for the generation of new antimicrobials to fight AMR infections. Despite this, most HDPs are produced by chemical synthesis, which is expensive, unsustainable, and difficult to scale-up. Alternatively, their recombinant production is very appealing but still challenging. HDPs are highly susceptible to degradation and are generally toxic to the recombinant host. However, inclusion bodies (IBs), which are protein aggregates that usually happen during recombinant production, can be used to allow HDP formation inside the host without being harmful. Also, the construction of chimeric proteins could be a strategy for successful recombinant expression of small peptides. In this context, this dissertation explores several new strategies for the recombinant production of HDPs. We tried leucine zippers as potential domains to drive the recombinant production of HDPs to the insoluble fraction and improve IBs protein quality. After that, we developed several antimicrobial multidomain proteins based on the fusion of different peptides and proteins from innate immunity. Because we also used leucine zippers with these constructs, they could be produced effectively – without toxicity to the microbial cell factory. Moreover, when needed, we were able to recover soluble antimicrobials from IBs using a mild, non-denaturing protocol. Overall, we demonstrated that these constructs have a broad-spectrum antimicrobial action against multi-drug resistant (MDR) bacteria, in both the soluble and IB format, and that they could trigger the release of IL-8 within a range of potential immunomodulatory properties. These outcomes invited us to use our constructs in the biofunctionalization of self-assembled monolayers to avoid biofilm formation. We observed that the chimeric proteins could be anchored to these materials and avoid biofilm growth. In sum, these results reinforce multidomain antimicrobial proteins as potential antimicrobial alternatives with immunomodulatory properties and open up the possibility for many applications of this new generation of antimicrobial protein nanoparticles as well as their soluble analogs.
8
Raposo, Renato Astolfi. "Análise de interações da subunidade catalítica da fosfatase do tipo 1 (PP1c) de Dictyostelium discoideum identificadas através do sistema de duplo-híbrido em leveduras." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-14052013-102905/.
Full textAbstract:
A proteína fosfatase do tipo-1 (PP1) é uma das principais proteínas serina/treonina fosfatases (PSTPs) e desempenha papeis fisiológicos tão diversos quanto importantes, tais como a regulação do metabolismo de carboidratos e do ciclo celular. A holoenzima PP1 é constituída por uma subunidade catalítica conservada (PP1c) que está associada a subunidades não-catalíticas que modulam sua localização subcelular, especificidade de substrato e atividade enzimática. Mais de 100 proteínas que interagem com a PP1c já foram identificadas em distintos organismos eucarióticos. Proteínas que interagem com a PP1c são, portanto, a chave para compreender os diferentes papéis biológicos da PP1. A subunidade catalítica da PP1 da ameba social Dictyostelium discoideum (DdPP1c) é codificada por um gene em cópia única o qual é expresso ao longo de todo o ciclo de vida desse organismo. Algumas proteínas que interagem e possivelmente modulam a atividade da PP1 de D. discoideum já foram identificadas, utilizando-se tanto buscas por similaridades na sequência genômica deste microorganismo como ensaios utilizando o sistema de duplo-híbrido em leveduras, utilizando-se a PP1c como isca. Com esta última abordagem, foram selecionados mais de 25 clones distintos de cDNA que codificam proteínas que potencialmente interagem com a DdPP1c, após varreduras de bibliotecas de cDNA de diferentes estágios de desenvolvimento de D. discoideum. Neste trabalho, nós confirmamos que o produto protéico de 11 destes clones interagem com a isca DdPP1c com base em novos ensaios de duplo-híbrido. Os demais clones codificam proteínas que não interagem com DdPP1c ou promovem auto-ativação do gene repórter. Selecionamos para estudos adicionais um clone do gene DDB_G0269300 cujo produto protéico predito de 423 de aminoácidos não tem função ainda conhecida. A sequência codificadora completa de DDB_G0269300 foi clonada para realização de novos ensaios de duplo-híbrido em leveduras, os quais confirmaram a especificidade de sua interação com DdPP1c. A proteína recombinante rDDB_G0269300 foi obtida com sucesso em bactérias, possibilitando a obtenção de anticorpos policlonais em camundongos. O anti-soro anti-rDDB_G0269300 é aparentemente específico no reconhecimento da proteína correspondente em extratos celulares de D. discoideum coletados em 12h e 16 da fase de desenvolvimento. Estes resultados coincidem com dados obtidos através de RT-qPCR que mostram aumento nos níveis dos transcritos de DDB_G0269300 entre 8h e 12h da fase de desenvolvimento, o que é indicativo da sua importância desta proteína durante esta fase do ciclo de vida de Dictyostelium como uma potencial parceira molecular da DdPP1cProtein phosphatase type-1 (PP1) is a major protein serine/threonine phosphatase (PSTP) which plays as diverse as important physiological roles, such as regulation of carbohydrate metabolism and of cell cycle. The PP1 holoenzyme comprises a conserved catalytic subunit (PP1c) associated with non-catalytic subunits that modulate its subcellular localization, substrate specificity and enzymatic activity. More than 100 proteins that interact with PP1c have been identified in different eukaryotic organisms. Therefore proteins that interact with PP1c are key to the understanding of PP1 different biological roles. The catalytic subunit of PP1 the social amoeba Dictyostelium discoideum (DdPP1c) is encoded by a single copy gene which is expressed throughout the life cycle of this organism. Some proteins that interact with and possibly modulate the activity of D. discoideum PP1 have been identified, using both similarity searches in the genome sequence of this microorganism as yeast two-hybrid screenings using PP1c as bait. With the latter approach, we have selected more than 25 distinct cDNA clones encoding proteins that potentially interact with DdPP1c after screening D. discoideum cDNA libraries from different developmental stages. In this study, we confirmed that the protein product from 11 of these clones interact with the bait DdPP1c based on two-hybrid assays. The other clones encode proteins that either does not interact or promote self-activation of the reporter gene. The clone related to DDB_G0269300 gene that encodes a predicted protein of 423 amino acids with unknown function was selected for further studies. DDB_G0269300 full-length coding sequence was cloned and new yeast two-hybrid assays were performed confirming the specificity of the interaction with DdPP1c. The recombinant protein rDDB_G0269300 was successfully obtained in bacteria and further used for polyclonal antibodies production in mice. The antiserum anti-rDDB_G0269300 is apparently specific for recognition of the corresponding protein in D. discoideum cell extracts collected after 12h and 16h of development. These results agree with RT-qPCR data showing that the levels of DDB_G0269300 transcripts are increased between 8 h and 12 h during the development, which is indicative of its importance during this phase in Dictyostelium life cycle as a DdPP1c potential molecular partner.
9
Comenale, Gabriela. "Expressão e purificação da proteína recombinante L2 do Papilomavírus bovino tipo-2 em sistema bacteriano." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/10/10132/tde-11102013-104819/.
Full textAbstract:
A papilomatose bovina é uma doença infectocontagiosa de ocorrência mundial, que assola o rebanho brasileiro, sem qualquer atitude efetiva de controle, e que tem como enfermidades associadas a tumores de bexiga hematúria enzoótica e tumores de trato digestório superior caraguatá, responsáveis por sensíveis perdas para a pecuária. Várias tentativas vacinais têm sido empreendidas com finalidades profiláticas ou terapêuticas, porém sem resultados eficazes. Esta situação se deve a aspectos relacionados à estrutura viral que dificultam uma manipulação eficiente para produção de produtos vacinais. Para que tais dados possam ser obtidos, faz-se necessário uma melhor compreensão da ação das proteínas recombinantes. A clonagem em vetores bacterianos para a expressão e purificação dessas proteínas serve a diferentes propósitos. Entre eles, a produção de insumos imunológicos, como testes diagnósticos ou mesmo vacinas. O presente projeto visou à expressão e purificação da proteína de capsídeo recombinante L2 de BPV-2. A proteína foi expressa em bactéria e tentou-se a purificação por coluna de afinidade; entretanto, problemas na purificação não possibilitaram a conclusão deste objetivo. Todas as abordagens e protocolos utilizados nesse trabalho são discutidos para contribuição ao conhecimento do processo.The bovine papillomatosis is an infectious disease of worldwide occurrence, plaguing the Brazilian herd, without any effective attitude control, and whose illnesses associated with bladder tumors \"enzootic hematuria\" and upper digestive tract tumors \"caraguatá\" sensitive responsible for losses to livestock. Several attempts have been undertaken vaccine with prophylactic or therapeutic purposes, but without effective results. This is due to issues related to viral structure that hinder efficient manipulation for production of vaccine products. In order to obtain such information, it is necessary better understanding of the action of recombinant proteins. The bacterial cloning vectors for the expression and purification of such proteins serve different purposes. Among them, the production of immune inputs, such as diagnostic tests or vaccines. This project aimed the expression and purification of recombinant L2 capsid protein of BPV-2. The protein was expressed in bacteria and purification was carried out by affinity column. However, difficulties in the purification process, impaired the full completion of this objective. All the attempted approaches and protocols were discussed and potential solutions proposed.
10
Teixeira, Lais Helena. "Geração e análise da imunogenicidade de proteínas recombinantes baseadas nas diferentes formas do antígeno circumsporozoíta de Plasmodium vivax visando o desenvolvimento de uma vacina universal contra malária." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/42/42133/tde-11072014-110149/.
Full textAbstract:
O P. vivax é a segunda espécie mais prevalente causadora de malária no mundo. Medidas de controle ineficientes exigem o desenvolvimento de novas estratégias de prevenção, como vacinas, novas drogas e novos inseticidas. O objetivo geral do trabalho foi gerar uma formulação vacinal universal com proteínas e adenovírus recombinantes capazes de induzir anticorpos contra as diferentes formas alélicas da proteína circumsporozoíta (CSP) do P. vivax. As proteínas foram produzidas em E. coli e purificadas por cromatografia de afinidade e troca iônica. A obtenção destas proteínas nos permitiu testar qual seria a melhor formulação vacinal para a indução de anticorpos contra as três formas alélicas da proteína CSP de P. vivax (PvCSP). Anticorpos específicos reconheceram esporozoítas do P. vivax por imunofluorescência. Por fim testamos o uso de dois adenovírus recombinantes, um símio e um humano, deficientes em replicação, expressando as três regiões imunodominantes da proteína PvCSP em fusão. Estes foram capazes de induzir resposta imune específica contra as proteínas PvCSP sendo testados em esquema de prime-boost heterólogo, onde camundongos foram primados com os adenovírus e nas doses-reforço receberam a mistura com as três proteínas recombinantes.The Plasmodium vivax is the second most prevalent species of malaria in the world. Inefficient measures of control used today demand the development of new strategies for prevention, as vaccines, new drugs and new insecticides. The central objective of this thesis was to generate a universal vaccine formulation with proteins and recombinant adenoviral vectors representing the different allelic forms of the circumsporozoite protein (CSP) of the P. vivax. The recombinant proteins were expressed in E. coli and purified. These proteins allowed us to test which would be the best vaccine formulation for the induction of antibodies against the three allelic forms of CSP. The specific antibodies also recognized P. vivax sporozoites by immunofluorescence. Finally we test the use of two recombinant adenoviral vectors, a simian and a human, both replication deficient, expressing a protein containing the repeat regions of the CSP in fusion. These adenoviral vectors induced specific immune response against CSP and were successfully used in an immunization regimen of heterologous prime and boost where in the first dose the mice received recombinant adenoviral vector and in the subsequent doses, the mixture with three recombinant proteins.
Books on the topic "ProteÃna recombinante"
1
Jean, Garnier, ed. Introduction to proteins and protein engineering. Amsterdam: Elsevier, 1988.
Find full text
2
Jean, Garnier, ed. Introduction to proteins and protein engineering. Amsterdam: Elsevier, 1986.
Find full text
3
Buckel, Peter, ed. Recombinant Protein Drugs. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7.
Full text
4
Tuan, Rocky S. Recombinant Protein Protocols. New Jersey: Humana Press, 1997. http://dx.doi.org/10.1385/089603481x.
Full text
5
MacDonald, Jacqueline, Igor Kolotilin, and Rima Menassa, eds. Recombinant Proteins from Plants. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3289-4.
Full text
6
Cunningham, Charles, and Andrew J. R. Porter, eds. Recombinant Proteins from Plants. Totowa, NJ: Humana Press, 1998. http://dx.doi.org/10.1007/978-1-60327-260-5.
Full text
7
Faye, Loïc, and Véronique Gomord, eds. Recombinant Proteins From Plants. Totowa, NJ: Humana Press, 2009. http://dx.doi.org/10.1007/978-1-59745-407-0.
Full text
8
Robson, Barry. Introductionto proteins and protein engineering. Amsterdam: Elsevier, 1988.
Find full text
9
Robson, Barry. Introduction to proteins and protein engineering. Amsterdam: Elsevier, 1986.
Find full text
10
Gasser, Brigitte, and Diethard Mattanovich, eds. Recombinant Protein Production in Yeast. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9024-5.
Full textBook chapters on the topic "ProteÃna recombinante"
1
Ge, Xumeng, and Jianfeng Xu. "Cell-Free Protein Synthesis as a Promising Expression System for Recombinant Proteins." In Recombinant Gene Expression, 565–78. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-433-9_30.
Full text
2
Weissmann, Charles. "Recombinant interferon - the 20th anniversary." In Recombinant Protein Drugs, 3–41. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_1.
Full text
3
Hofschneider, Peter Hans, and Kenneth Murray. "Combining science and business: from recombinant DNA to vaccines against hepatitis B virus." In Recombinant Protein Drugs, 43–64. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_2.
Full text
4
Brownlee, George G., and Paul L. F. Giangrande. "Clotting factors VIII and IX." In Recombinant Protein Drugs, 67–88. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_3.
Full text
5
Welte, Karl, and Erich Platzer. "Colony-stimulating factors: altering the practice of oncology." In Recombinant Protein Drugs, 89–106. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_4.
Full text
6
Collen, Désiré, and H. Roger Lijnen. "Tissue-type plasminogen activator: helping patients with acute myocardial infarction." In Recombinant Protein Drugs, 107–26. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_5.
Full text
7
Gillies, Stephen D. "Designing immunocytokines: genetically engineered fusion proteins for targeted immune therapy." In Recombinant Protein Drugs, 129–47. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_6.
Full text
8
Burke, Paul A., and Scott D. Putney. "Improving protein therapeutics: the evolution of the modern pharmacopoeia." In Recombinant Protein Drugs, 151–68. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_7.
Full text
9
Friedmann, Theodore. "Principles of gene transfer and foreign protein expression for human gene therapy." In Recombinant Protein Drugs, 169–80. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_8.
Full text
10
Micsonai, András, Éva Bulyáki, and József Kardos. "BeStSel: From Secondary Structure Analysis to Protein Fold Prediction by Circular Dichroism Spectroscopy." In Methods in Molecular Biology, 175–89. New York, NY: Springer US, 2020. http://dx.doi.org/10.1007/978-1-0716-0892-0_11.
Full textAbstract:
Abstract Far-UV circular dichroism (CD) spectroscopy is a classical method for the study of the secondary structure of polypeptides in solution. It has been the general view that the α-helix content can be estimated accurately from the CD spectra. However, the technique was less reliable to estimate the β-sheet contents as a consequence of the structural variety of the β-sheets, which is reflected in a large spectral diversity of the CD spectra of proteins containing this secondary structure component. By taking into account the parallel or antiparallel orientation and the twist of the β-sheets, the Beta Structure Selection (BeStSel) method provides an improved β-structure determination and its performance is more accurate for any of the secondary structure types compared to previous CD spectrum analysis algorithms. Moreover, BeStSel provides extra information on the orientation and twist of the β-sheets which is sufficient for the prediction of the protein fold. The advantage of CD spectroscopy is that it is a fast and inexpensive technique with easy data processing which can be used in a wide protein concentration range and under various buffer conditions. It is especially useful when the atomic resolution structure is not available, such as the case of protein aggregates, membrane proteins or natively disordered chains, for studying conformational transitions, testing the effect of the environmental conditions on the protein structure, for verifying the correct fold of recombinant proteins in every scientific fields working on proteins from basic protein science to biotechnology and pharmaceutical industry. Here, we provide a brief step-by-step guide to record the CD spectra of proteins and their analysis with the BeStSel method.
Conference papers on the topic "ProteÃna recombinante"
1
Berkner, K. L., S. J. Busby, J. Gambee, and A. Kumar. "EXPRESSION IN MAMMALIAN CELLS OF FUSION PROTEINS BETWEEN HUMAN FACTORS IX AND VII." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643568.
Full textAbstract:
The vitamin K-dependent plasma proteins demonstrate remarkable similarities in their structures: all have multiple domains in common and extensive homology is observed within many of these domains. In order to investigate the structure-function relationship of these proteins, we have interchanged domains of one protein (factor IX) with that of another (factor VII) and have compared the expression of these fusion proteins with recombinant and native factors IX and VII. Oligonucleotide-directed mutagenesis was used to generate four fusion proteins: factor IX/VII-1, which contains the factor IX leader and gla domain fused to the growth factor and serine protease of factor VII; factor VII/IX-1, a reciprocal fusion protein of factor IX/VII-1; factor IX/VII-2, which contains the factor IX leader adjoined to the mature factor VII protein sequence; and factor VII/IX-2, the reciprocal fusion protein of factor IX/VII-2. The cDNAs encoding all four proteins were cloned into mammalian expression vectors, and to date three of these (factors IX/VII-1, 2 and VII/IX-1) have been transfected into baby hamster kidney (BHK) cells or 293 cells and characterized. Factors IX/VII-1 and VII/IX-1 were both secreted at levels comparable to recombinant factors IX and VII. The factor IX/VII-1 was identical in molecular weight to native or recombinant factor VII (i.e., 53 K). Factor VII/IX-1 was expressed as two proteins with molecular weights around 68 kd, as observed with recombinant factor IX. The factor IX/VII-1 protein has been purified to homogeneity and has been found to possess factor VII biological activity, but at a specific activity approximately 20% that of plasma factor VII. Thus, the gla domain of one clotting factor is capable of directing the activation of another and of generating biologically active protein. In contrast, no activity was observed with the factor IX/VII-2 fusion protein, indicating that there are limits to the interchanges which can generate functional blood clotting factors.
2
Tyumentsev, A. I., M. A. Tyumentseva, and V. G. Akimkin. "DEVELOPMENT OF APPROACHES FOR ENDOTOXIN REMOVAL FROM PROTEIN PREPARATIONS ON THE EXAMPLE OF NUCLEASES OF THE CRISPR/CAS SYSTEM." In Molecular Diagnostics and Biosafety. Federal Budget Institute of Science 'Central Research Institute for Epidemiology', 2020. http://dx.doi.org/10.36233/978-5-9900432-9-9-113.
Full textAbstract:
Removal of bacterial endotoxins from solutions of recombinant proteins is one of the most important issues in the preparation of highly purified preparations suitable for in vivo use. An optimal technology for obtaining preparations purified from bacterial endotoxins has been proposed using purification of preparations of recombinant nucleases of the CRISPR/CAS system as an example. Efficacy of developed technology was compared with other available methods. Removal of bacterial endotoxins was carried out using Triton X-114 detergent added to a concentration of 1% to a solution containing the recombinant protein. It was shown that the content of bacterial endotoxins in solutions of purified proteins obtained according to the proposed technology is 0.3–1.5 EU/ml.
3
Vehar, G. A. "THE PRESENT STATE OF GENE TECHNOLOGY IN THE MANUFACTURE OF HUMAN COAGULATION PROTEINS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644755.
Full textAbstract:
The production of pharmaceuticals from human plasma that are useful in the treatment of bleeding disorders had its beginning with the development of the Cohn fractionation procedure in the 1940's. As a result of these advances, concentrates became available for the treatment of the hemophilias. Although of low purity and subject to contamination by hepatitis virus, the availability of these compounds resulted in dramatic improvements in the life expectancy and quality of life of afflicted individuals. The numerous problems associated with production of pharmaceuticals from pooled plasma made these products obvious goals for recombinant DNA technology as soon as the commercial aspects of the field became apparent. The subsequent contamination of blood products with the AIDS virus has resulted in an urgent need for a production source that is independent of human plasma. Several industrial and academic laboratories have cloned the cDNA's for human factors VIII and IX. In addition to these proteins, the utility of factor Vila in the treatment of hemophiliacs with inhibitors has shown promise. Efforts to develop a recombinant preparation of factor Vila are at a comparable stage of development as factors VIII and IX. Continuing efforts have resulted in the successful expression of these recombinant proteins in mammalian cell lines, thereby successfully completing the first steps of commercial development.Although much interest has focused upon the theoretical superiority of recombinant proteins as therapeutics, one must keep in mind that there are numerous developmental aspects of large-scale production and regulatory issues that must be addressed and solved before these drugs will be available. The coagulation proteins are complex glycoproteins that will in all probability require mammalian cell cell culture in order to produce functional proteins. The fact that these preparations will be administered over the lifetime of the patient serves to reinforce that the recombinant products be as similar to the natural proteins as possible, further supporting the concept of mammalian cell expression systems.Regulatory approval of a recombinant product are fundamentally no different than those for any other product in regards to efficacy, potency, purity, and identity. There are, however, additional considerations that must be addressed in the production of recombinant cell culture derived biologies. These relate to the possible presence in the final product of pathogenic and tumorigenic agents, and possible contamination by cell culture and cell substrate compounds. A detailed characterization of the production cell line will therefore be required, including identification and characterization of any associated viral particles. These cells must be capable of being reproducibly grown, while maintaining protein production, on ascale (tens of thousands of liters) suitable to meet the market demand of the specific protein. Apurification process must be established capable of handling the resulting large volumes of feedstock, generating a protein preparation of high purity (greater than 99% pure). Numerous assays must be developed to quantitate the purity and identity of the resulting recombinant pharmaceutical on a lot by lot basis.Studies to date have shown that recombinant forms of factors VIII and IX, produced by laboratory processes, are very similar to the plasma-derived forms as assessed by a variety of in vitro and in vivo tests. Although these results are promising, the ultimate safety and efficacy testing of these drugs will have to await the initiation of human clinical trials. Such studies will have to await the successful completion of the certain regulatory concerns. Clinical trials should begin within the near future, hopefully leading to a source of these products independent of pooled human plasma.
4
Ashok Kumar, A., Margaret Insley, Jay Gambee, Sharon J. Busby, and Kathleen L. Berkner. "SITE SPECIFIC MUTAGENESIS WITHIN THE GLA-DOMAIN OF HUMAN FACTOR IX." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644079.
Full textAbstract:
Factor IX, a plasma protein, plays a critical role in blood coagulation. The biological activity of factor IX as well as several other plasma proteins depends on the presence of gamma-carboxy glutamic acid (Gla) residues in their amino terminal region. In vitro mutagenesis has been used to selectively replace Gla residues of factor IX with aspartic acid (Asp) residues in order to establish the contribution of individual as well as paired Gla residues to the normal functioning of the protein. These substitutions were made at positions 7, 15, 20 and 26 in human factor IX. In addition, residue number 18, a cysteine has been changed to serine in an attempt to disrupt the highly conserved disulfide bond in the gla-domain. The gla-domain mutants will be produced in mammalian cells and compared with native recombinant factor IX. A rapid immunoaffinity purification procedure, which has been used to obtain recombinant factor IX produced in the presence or absence of vitamin K, is being used to purify the mutants. Protein sequence analysis has been used to confirm complete processing and proper gamma-carboxylation of recombinant factor IX. The properties of these mutants as compared to human factor IX will be discussed.
5
Teng, Weibing, Yiding Huang, Joseph Cappello, and Xiaoyi Wu. "Mechanical and In-Vitro Cell Compatibility Properties of Silk-Elastinlike Protein-Based Biomaterial." In ASME 2010 First Global Congress on NanoEngineering for Medicine and Biology. ASMEDC, 2010. http://dx.doi.org/10.1115/nemb2010-13141.
Full textAbstract:
A series of genetically engineered recombinant silk-elastinlike proteins (SELPs) have been produced by combining polypeptide sequences derived from native silk of superior mechanical strength and elastin that is extremely durable and resilient. They have displayed a set of outstanding properties such as good biocompatibility and controllable biodegradation rates. In the study, we characterized the mechanical property of genetically engineered, recombinant silk-elastinlike protein copolymer, SELP-47K, under physical and chemical treatments. The biocompatibility of the SELP-47K was also evaluated by cell culture. The ultimate goal of this study is to explore the potential of SELPs for applications in the engineering of load-bearing tissues such as arteries.
6
Furis, B. C., M. J. Jorgensem, M. J. Rabiet, A. B. Contor, C. L. Brown, C. B. Shoemaker, and B. Furie. "RECOGNITION SITE DIRECTING GAMMA-CARBOXYLATION RESIDES ON THE PROPEPTIDES OF FACTOR IX AND PROTRROMBIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643992.
Full textAbstract:
Factor IX and prothrombin vitamin K-dependent proteins that participate in blood coagulation undergo post-translationalmodification in which glutamic acid residues in the amino terminus of the protein are converted to gamma-carboxyglutamic acid residues. This modification confers divalent metal ion binding ability upon the proteins.As a consequence of binding divalent metal ions these proteins undergoconformational changes necessary for biological function.The vitamin K-dependent proteins are synthesized with an NH2-terminal extension. The region distal to the NH2-terminus of the mature protein is a prototypic signal sequence while the proximal region is a propeptide with homology among the vitamin K-dependent proteins. The boundary between the pre and pro sequences has been established for factor IX by analysis of three naturally occurring factor IX mutants factor IX Cambridge factor IX Oxford-3 and factor IX San Dimas, in which processing is incomplete.For human factor IX the propeptide extends from residue -18 to -1. The homology among the propeptides of vitamin K-dependent proteins suggests that the propeptide may designate adjacent gamma-carboxyglutamic acids for carboxylation. To test this hypothesis alterations in sequence were introduced into the propeptide region of human factor IX cDNA by oligonucleotide directed site specific mutagenesis.Mutated genes were expressed in Chinese hamster ovary cells. Rapid and efficient isolationof the mutant proteins by immunoaffinity chromatography permitted detailed analysis of the mutants on quantities of protein easily obtainable at low expression levels. The extent of gamma-carboxylation was assessed by the ability of the mutant proteins to interact with conformation specific antibodies directed against the gamma-carboxyglutamic acid-dependent metal stabilized native structure of factor IX as well as by direct amino acid analysis. Unmodified recombinant factor IX contained, on average, 9 gamma-carboxyglutamic acid residues, as compared to 12 for plasma factor IX. About 70% of the recombinant wild type factor IX bound to the conformation specific antibodies. Deletion of the propiece or point mutations at residues -10 or -16 led to secretion of uncarboxylated factor IX unreaotive with antibodies specific for the native structure but with the NH2-terminus of mature factor IX. In order to assess the universality of these observations we have recently cloned human prothrombin cDNA and expressed the gene in the same Chinese hamster ovary cell system used for factor IX. In contrast to factor IX, at low levels ofexpressionof the prothrombin gene, the prothrombin is fully carboxylated relative to a plasma prothrombin standard.The recombinant prothrombin exhibits the same specific clotting activity as plasma derivedprothrombin and is fully native as evaluated by conformation specific antibodies. At high levels of expression the capacityof the cells to carboxylate prothrombin can be exceeded leading to secretion of under carboxylated prothrombin. However, the absolute amount of fully carboxylated prothrombin that can be produced in this system appears to be a least fivefold greater that the absolute amount of highly carboxylated factor IX that can be synthesized.The elimination of carboxylation observed upon mutation of the propiece of factor IX suggest that the propiece contains a recognition element required for carboxylation of the protein. Assignment of a functional role to the propiece of factor IX represents the first determination of function for any pro sequence. It is anticipated that extension of these studies to prothrombin will demonstrate that this recognition signal is used by all the members of this class of proteins. In order to determine if the propiece is sufficient to designate a protein for gamma-carboxylation we are currently constructing chimeric proteins incorporating the propieceof prothrombin into the cDNA of normally uncarboxylated proteins.
7
Novikova, L. I., S. S. Bochkareva, A. V. Aleshkin, S. IU Kombarova, O. E. Karpov, A. A. Pulin, O. A. Orlova, IU S. Lebedin, A. M. Vorobev, and E. R. Mekhtiev. "DYNAMICS OF ANTIBODIES TO VARIOUS ANTIGENS OF THE SARS-COV-2 CORONAVIRUS IN PATIENTS WITH CONFIRMED COVID-19 INFECTION." In Molecular Diagnostics and Biosafety. Federal Budget Institute of Science 'Central Research Institute for Epidemiology', 2020. http://dx.doi.org/10.36233/978-5-9900432-9-9-159.
Full textAbstract:
Presence of IgG and IgM antibodies in venous blood of 76 patients with confirmed presence of SARS-CoV-2 was determined. The study was carried out by ELISA using Russian test systems. Revealed different levels of IgM antibodies to N-protein and RBD (receptor binding domain of the Spike protein). The level of IgM to RBD did not reach high values, while the level of IgM to N-protein sharply increased in a short period of time to high values by the 3rd week of the disease and decreased only by the 8th week. The dynamics of IgG antibodies to the whole virion antigen and the recombinant spikes was similar, reaching high values at 4-5 weeks of the disease, however, the dynamics of IgG to the N-protein differed, showing a slight increase by the 1st week of the disease and a low level throughout the entire period of observation. Different dynamics of production of IgG and IgM antibodies to N-protein and RBD were noted. The amount of IgM to the N-protein increased sharply and reached a high level, while the amount of IgG increased smoothly and did not show a high level. The opposite picture was observed for antibodies to RBD. The characteristic dynamics of IgG, measured using test systems withsorbed whole virion or recombinant spike proteins, suggests duration of the disease
8
Qiu, Weiguo, Arjun Stokes, Joseph Cappello, and Xiaoyi Wu. "Electrospinning of Recombinant Protein Polymer Nanofibers." In ASME 2009 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2009. http://dx.doi.org/10.1115/sbc2009-206352.
Full textAbstract:
Structural proteins often in the form of micro and nanofibers, constituting most of intra- and extracellular matrix (ECM), are the fundamental building blocks of life [1]. Recent efforts to replace diseased or damaged tissues and organs have resulted in the molecular design and genetic engineering of recombinant proteins, and the advent of new technology for fabricating structural proteins into micro-/nanofibrous scaffolds, hoping to resemble some or all the characteristics of ECM structure and function. The fabrication of such an ECM mimic may be an important step in engineering a functional tissue. To this end, we have produced a series of silk-elastin-like proteins (SELPs) [2]. Revealed by our subsequent studies, SELPs in the form of hydrogels, thin films, and microfibers, have displayed a set of outstanding biological and physical properties. In this study, electrospinning will be pursued as a mechanism for the formation of SELP nanofibers.
9
Qiu, Weiguo, Joseph Cappello, and Xiaoyi Wu. "Fabrication of Genetically Engineered Silk-Elastin-Like Protein Polymer Fibers." In ASME 2008 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2008. http://dx.doi.org/10.1115/sbc2008-190980.
Full textAbstract:
Micro- and submicro-diameter protein fibers are fundamental building blocks of extra- and intra-cellular matrices, providing structural support, stability and protection to cells, tissues and organism [1]. Fabricating performance fibers of both naturally derived and genetically engineered proteins has been extensively pursued for a variety of biomedical applications, including tissue engineering and drug delivery [2]. Silk-elastin-like proteins (SELPs), consisting of tandemly repeated polypeptide sequences derived from silk and elastin, have been biosynthesized using recombinant DNA technique [3]. Their potential as a biomaterials in the form of hydrogels continues to be explored [4, 5]. This study will focus on the fabrication of robust, micro-diameter SELP fibers as biomaterials for tissue engineering applications.
10
Verbeet, M. Ph, R. F. Evers, A. Leyte, H. L. Lamain, A. J. J. Van Ooyen, J. A. Van Mourik, and H. Pannekoek. "DETERMINATION OF THE DOMAINS OF FACTOR VIII ESSENTIAL FOR PROCOAGULANT ACTIVITY." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643613.
Full textAbstract:
Factor VIII (FVIII) consists of an obvious domain structure that can be represented as A1-A2-B-A3-C1-C2 (Vehar et al., 1984, Nature 312, 337). In order to determine the domains involved in the procoagulant activity of FVIII, we constructed mutant FVIII cDNAs containing deletions in the coding sequence of the full-length molecule. In one of the mutants a large part of the B domain is deleted. In another one we made a deletion in the B domain that extends beyond the thrombine cleavage site. We used pSV-2 derived expression vectors and COS-1 cells in a transient expression system for the full-length and mutant recombinant proteins. Conditioned media (CM) were harvested.In accordance with the described mutants of recombinant FVIII (Toole et al., 1986, PNAS 83, 5939), we demonstrated an increase in activity in the CM for these mutants as compared to the full-length activity. We also found that the specific activity of the mutants is similar to that of plasma FVIII. So, shorter chains lead to an increased amount of procoagulant protein.
Reports on the topic "ProteÃna recombinante"
1
Rueda, Paloma. Proteínas recombinantes, ¿cómo y para qué? VLPs: un ejemplo interesante. Sociedad Española de Bioquímica y Biología Molecular (SEBBM), July 2013. http://dx.doi.org/10.18567/sebbmdiv_rpc.2013.07.1.
Full text
2
Veen, Ryan Vander, Mark Mogler, Matthew M. Erdman, and D. L. Hank Harris. Preparation of GP5-M Heterodimer Glycantype Specific Recombinant Protein and Replicon Particles. Ames (Iowa): Iowa State University, January 2009. http://dx.doi.org/10.31274/ans_air-180814-698.
Full text
3
Walls, Lichun H. Isolation and Preliminary Characterization of a Recombinant TAT Protein From Human Immunodeficiency Virus. Fort Belvoir, VA: Defense Technical Information Center, May 1995. http://dx.doi.org/10.21236/ada298304.
Full text
4
Adams, Michael W., and Michael W. W. Adams. MAGGIE Component 1: Identification and Purification of Native and Recombinant Multiprotein Complexes and Modified Proteins from Pyrococcus furiosus. Office of Scientific and Technical Information (OSTI), January 2014. http://dx.doi.org/10.2172/1113776.
Full text
5
Cowens, J. W., and M. J. Ehrke. Application of Laboratory Robotics to the Determination of the Primary Structure of Recombinant Proteins and the Measurement of Endotoxin. Fort Belvoir, VA: Defense Technical Information Center, September 1994. http://dx.doi.org/10.21236/ada294245.
Full text
6
Chan, Eva. Expression and Purification of Recombinant Protein to Generate a Monoclonal Antibody to the PX domain of Tks5 ? Isoform in Cancer Cells. Portland State University Library, January 2016. http://dx.doi.org/10.15760/honors.323.
Full text
7
Angov, Evelina. Production of a Recombinant E. coli Expressed Malarial Vaccine from the C-Terminal Fragment of Plasmodium Falciparum 3D7 Merozoite Surface Protein-1. Fort Belvoir, VA: Defense Technical Information Center, September 2000. http://dx.doi.org/10.21236/ada391249.
Full text
8
THE RECOMBINANT PROTEIN CYTOTOXIC NECROTIZING FACTOR FROM STRAIN YERSINIA PSEUDOTUBERCULOSIS, ISOLATED IN THE RUSSIAN FEDERATION. НИИ эпидемиологии и микробиологии им. Г.П. Сомова, 2016. http://dx.doi.org/10.18411/hmes.d-2016-075.
Full text