Academic literature on the topic 'Recombinant protein'
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 'Recombinant protein.'
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 "Recombinant protein"
1
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.
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
Lewis, Peter. "Recombinant protein drugs." British Journal of Clinical Pharmacology 53, no. 4 (April 2002): 411. http://dx.doi.org/10.1046/j.1365-2125.2002.01571.x.
Full text
4
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.
5
Rodrigues, M., S. Li, K. Murata, D. Rodriguez, J. R. Rodriguez, I. Bacik, J. R. Bennink, J. W. Yewdell, A. Garcia-Sastre, and R. S. Nussenzweig. "Influenza and vaccinia viruses expressing malaria CD8+ T and B cell epitopes. Comparison of their immunogenicity and capacity to induce protective immunity." Journal of Immunology 153, no. 10 (November 15, 1994): 4636–48. http://dx.doi.org/10.4049/jimmunol.153.10.4636.
Full textAbstract:
Abstract We compared the effectiveness of several recombinant influenza and vaccinia viruses to induce a malaria-specific immune response. The CD8+ T cell epitope of the circumsporozoite (CS) protein of Plasmodium yoelii, a rodent malaria parasite, was expressed in two distinct influenza virus proteins, the hemagglutinin and the neuraminidase. These recombinant viruses were found to be equally efficient at inducing CS-specific CD8+ T cells in mice. A third recombinant virus, which expresses a B cell epitope of the CS protein, induced neutralizing anti-sporozoite Abs. Expression in the same recombinant virus of the CD8+ T cell epitope and of the B cell epitope did not impair the capacity of this recombinant virus to induce malaria-specific CD8+ T cells and neutralizing Abs. The immunogenicity of a vaccinia virus, expressing the entire CS protein, was compared with that of a highly attenuated vaccinia strain expressing the same protein and with that of another vaccinia virus expressing only the CD8+ T cell epitope. All three vaccinia virus recombinants elicited CS-specific CD8+ cells and a potent inhibitory response against pre-erythrocytic stages of malaria parasites. Optimal levels of anti-sporozoite Abs, inhibition of liver stage development, and protection against malaria infection resulted from repeatedly immunizing the animals with recombinant influenza viruses followed by boosters with a recombinant vaccinia virus. These findings support the concept that live viral vectors expressing the appropriate proteins and/or epitopes can be used as promising vaccine candidates.
6
Llompart, Blanca, Immaculada Llop-Tous, Pablo Marzabal, Margarita Torrent, Roser Pallissé, Miriam Bastida, M. Dolors Ludevid, and Fabien Walas. "Protein production from recombinant protein bodies." Process Biochemistry 45, no. 11 (November 2010): 1816–20. http://dx.doi.org/10.1016/j.procbio.2010.01.016.
Full text
7
Lobb, Leslie, Boguslaw Stec, Evan K. Kantrowitz, Akihito Yamano, Vivian Stojanoff, Ofer Markman, and Martha M. Teeter. "Expression, purification and characterization of recombinant crambin." "Protein Engineering, Design and Selection" 9, no. 12 (1996): 1233–39. http://dx.doi.org/10.1093/protein/9.12.1233.
Full text
8
Ohtake, Satoshi, and Tsutomu Arakawa. "Recombinant Therapeutic Protein Vaccines." Protein & Peptide Letters 20, no. 12 (November 2013): 1324–44. http://dx.doi.org/10.2174/092986652012131112122245.
Full text
9
Fuerst, Walter. "Recombinant Activated Protein C." Critical Care Medicine 32, no. 1 (January 2004): 311–12. http://dx.doi.org/10.1097/01.ccm.0000104932.57061.ad.
Full text
10
Stein, M.D.,, Richard A. "Recombinant Protein Expression Advances." Genetic Engineering & Biotechnology News 31, no. 16 (September 15, 2011): 34–36. http://dx.doi.org/10.1089/gen.31.16.14.
Full textDissertations / Theses on the topic "Recombinant protein"
1
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
2
Tse, Muk-hei. "Investigations on recombinant Arabidopsis acyl-coenzyme A binding protein 1." View the Table of Contents & Abstract, 2005. http://sunzi.lib.hku.hk/hkuto/record/B36427664.
Full text
3
Tse, Muk-hei, and 謝牧熙. "Investigations on recombinant Arabidopsis acyl-coenzyme A binding protein 1." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B36427664.
Full text
4
Corgozinho, Carolina Nunes Costa. "Desenvolvimento de vacina baseada em sistema de liberação sustentada contendo proteína recombinante." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/60/60137/tde-31072009-083709/.
Full textAbstract:
No Brasil, e em outros paises de clima tropical, os carrapatos se tornaram um enorme problema economico de^$de que a industria do gado se desenvolveu. O carrapato Boophilus microplus, um dos artropodes mais importantes na veterinaria, causa efeitos direto, como suc,cao de sangue, e indireto, como a transmissao de uma grande variedade de patogenos que normalmente resulta em infec,c~es letais. As vacinas genicas contendo o antigeno Bm86, uma proteina ligada a membrana do intestino do carrapato B. microplus, representam uma alternativa atrativa aos acaricidas para controlar as infesta~oes por carrapatos em contrapartida aos inconvenientes produtos quimicos. Devido sua administra,cao ser feita em 4 doses no primeiro ano, seguida de refor,cos a cada seis meses, estas formula,coes vacinais nao s3c adequadas para paises com cria,cao extensiva de gado, como no Brasil. Visando uma libera~ao sustentada do antigeno Bm86, neste trabalho desenvolveuse uma vacina de dose unica baseada em microesferas polimericas. Para obter o padrao de liberac,ao desejavel, diferentes formula,coes e parametros de processo foram variados, como a composi,cao do polimero, a taxa entre os monomeros ^Uacido latico:acido glicolico\" e o tamanho das microparticulas. As formula,coes foram preparadas pelo metodo de emulsao multipla e evapora,cao do solvente. A formula~ao que melhor se enquadrou nos objetivos da vacina de dose unica foi preparada com PLGA 75:25, solu,cao 3% de PVA como estabilizante, agita,cao de 11000 rpm para forma,cao da emulsao primaria e de 800 rpm para forma,cao da emulsao multipla e evapora,cao do solvente. As particulas assim obtidas apresentaram um tamanho medio de 25 ,um, uma taxa de encapsula,cao maior que 90% e aproximadamente 50% da proteina foi liberada in vitro em 60 dias. Analises por SDS-PAGE e Westem Bloning revelaram que a proteina se manteve integra apos encapsula,cao. Os resultados da avalia,cao da imunogenicidade em bovinos mostraram que a formula,cao baseada em microesferas polimericas biodegradaveis e habil a conseguir, com uma unica dose, uma resposta imune similar aquela conseguida com tres doses das formula,coes convencionais da vacina de Bm86.In Brazil, and in others tropical countries, the ticks have become a huge economic problem since the industry of livestock has developed. Ticks and tick-borne diseases affect animal and human health and are the cause of significant economic losses. The cattle tick Boophilus microplus is one of the most important arthropods in veterinary. This tick species causes both direct effects, such as blood sucking, and indirect effects, such as transmission of a wide variety of pathogens, which usually result in lethal infections. The gene vaccines based on Bm86 antigen, a midgut membrane-bound protein of the cattle tick B. microplus, represent a good alternative to control tick infestations, compared to chemicals. However, due to these vaccine formulations need 4 doses over the first year with booster at each 6 months to be effective, they are not suitable for countries with extensive cattle raising, like Brazil. Aiming a sustained release of Bm86 antigen, in this work we developed a single shot vaccine based on Bm86 loaded polymeric microspheres. In order to obtain desired release patterns, different formulations and processing parameters were varied, for example, the composition of the polymer, the monomer ratio lactic acid:glycolic acid and the size of the microparticles. The formulations were prepared by solvent evaporation method based on double emulsion. The formulation that presented better result as single shot vaccine was prepared with PLGA 75:25, solution 3% of PVA as stabilizer, agitation of 11000 rpm to form the primary emulsion and 800 rpm to obtain the double emulsion and solvent evaporation. The particles thus obtained presented an average size of 25 m, encapsulation ratio greater than 90% and approximately 50% of the protein was released in vitro in 60 days. Analysis by SDSPAGE and Western Blot showed that the integrity of the protein remained after encapsulation. The immunogenic studies showed that the formulation based onbiodegradable polymeric microspheres is able to elicit, with a single dose, an immune response and protection similar to that attained with 3 doses of conventional Bm86 vaccine formulations.
5
Ndabambi, Nonkululeko. "Recombinant expression of the pRb- and p53-interacting domains from the human RBBP6 protein for in vitro binding studies." Thesis, University of the Western Cape, 2004. http://etd.uwc.ac.za/index.php?module=etd&.
Full textAbstract:
The aim of this thesis was to produce DNA expression constructs and use them to investigate the feasibility of recombinantly expression proteins for future interaction studies between human RBBP6 and p53 and pRb proteins.
6
Barua, Bipasha. "Design and study of Trp-cage miniproteins /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/8533.
Full text
7
Bleckwenn, Nicole Aleece. "Protein production development with recombinant vaccinia virus." College Park, Md. : University of Maryland, 2004. http://hdl.handle.net/1903/1416.
Full textAbstract:
Thesis (Ph. D.) -- University of Maryland, College Park, 2004.Thesis research directed by: Chemical Engineering. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
8
Morreale, Giacomo. "Processing of recombinant fusion protein and peptide." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615060.
Full text
9
Pesarrodona, Roches Mireia. "Supramolecular organisation and biological properties of tumor targeted, self-assembling protein nanoparticles." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/402365.
Full textAbstract:
Degut a la seva flexibilitat funcional i arquitectònica, l’ús de proteïnes recombinants formades per múltiples dominis representen una eina interesant pel desenvolupament de nanopartícules capaces de transportar fàrmacs de manera específica. Recentment, en el nostre grup, hem aplicat un nou sistema d’enginyeria pel desenvolupament de nanoparticles formades mitjançant l’oligomerització de proteïnes modulars. L’eficàcia selectiva i l’absència de toxicitat d’unes nanopartícules dirigides al càncer colorectal demostra la capacitat d’aquest enfoc biotecnològic. Un enfoc basat en l’ús de pèptids catiònics en els extrems terminal d’una proteïna modular que li confereix la capacitat d’interaccionar amb sí mateixa formant nanopartícules proteiques. Per tal d’explorar la flexibilitat d’aquest enfoc, hem desenvolupat dues nanopartícules proteiques, anomenades A5G27-GFP-H6 i FNI/II/V-GFP-H6, que reconeixen i internalitzen, de manera específica, cèl·lules que expressen el receptor CD44. Aquestes proteïnes modulars formen estructures anulars de 14 nm, estables en plasma capaces d’internalitzar a través d’endocitosis sense presentar toxicitat cel·lular. Aquestes nanopartícules específiques són una plataforma prometedora com a nanomedicines pel transport de fàrmacs o sondes pel diagnòstic i tractament d’afliccions relacionades amb CD44.
Durant la producció recombinant microbiana, moltes proteïnes heteròlogues tendeixen a acumular-se en cossos d’inclusió. Tenint en compte la última descripció d’aquestes entitats, constitueixen una font de proteïna pràcticament pura. Tot i que s’han desenvolupat tècniques per l’obtenció de proteïna activa mitjançant l’ús de detergents solubilitzadors no desnaturalitzants de manera satisfactòria, és desconegut com aquests procediments afecten el procés d’oligomerització de nanopartícules proteiques. En aquesta tesi, hem analitzat quin impacte té, a nivell estructural i funcional, l’origen de la font proteica. Depenent de si les nanopartícules deriven de la fracció soluble o de la resolubilització dels cossos d’inclusió, s’obtenen diferents organitzacions supramoleculars amb comportaments funcionals diferenciats. Aquests resultats demostren la rellevància que té l’origen cel·lular del material quan parlem d’estructures proteiques complexes.
Tot i que recentment s’ha demostrat que la bactèria hoste té una influencia directe en la estructura i el rendiment de les nanopartícules proteiques, es desconeix com els hi afecta l’ús de bactèries amb un perfil genètic diferent com soques deficients en xaperones o lliures de lipopolisacàrids. En aquesta tesi, s’ha investigat l’estructura de nanopartícules dirigides a CD44 o CXCR4 produïdes en diferents soques d’E.coli i com les característiques fisicoquímiques afecten la biointeracció d’aquestes nanopartícules. Els resultats demostres la robustesa dels patrons d’oligomerització d’aquestes nanopartícules dirigides a tumor i la influencia que té el perfil genètic microbià en la proporció de les diferents poblacions oligomèriques en el producte cru. Aquesta variabilitat, intrínseca de la bioproducció, tot i ser imprevisible o incontrolable, constitueix un ventall de nanomaterial. De manera exclusiva es pot seleccionar aquella població de nanopartícules amb les característiques funcionals i fisicoquímiques òptimes considerant l’aplicació per la qual s’han dissenyat.Recombinant multidomain protein building-blocks have demonstrated to be an appealing biotechnological approach for the development of cell-targeted, nanoscale drug carriers on account of protein functional and architectonic versatility. The colorectal tumor targeting efficacy as well as non-toxicity of T22-empowered nanoparticles validates the potential of the de novo engineering approach developed in our research group. An approach based on end-terminal cationic peptides as pleiotropic tags in self-assembling protein building blocks. In an attempt to explore the flexibility of this approach, we have developed two protein-only nanoparticles that specifically bind CD44-receptor, namely A5G27-GFP-H6 and FNI/II/V-GFP-H6. These designed building-blocks promote the formation of ring-shaped structures which are approximately 14 nm in size, stable in plasma that can internalise cells through endocytosis in absence of cell toxicity. These targeted protein nanoparticles, together with the stability and biodistribution of other tested multifunctional self-assembling protein nanoparticles, are promising platforms for the transport of drugs or imaging agents as nanomedicines for breast cancer or other CD44-linked afflictions. Referring to microbial cell factories, many of the recombinant proteins are likely accumulated in IBs upon induction stress; however, considering non-classical structure description of these protein build-ups they represent an abundant and pure protein source. Although active protein has been obtained from IBs through mild solubilisation procedures, it is still unknown how the recovery of active protein from IBs affects the self-assembling process into protein nanoparticles. Thus, we have analyse the impact on nanoparticle structure and functionality considering the protein material source (nanoparticles from the soluble cell fraction or resolubilised from IBs) of the developed CD44-targeted protein vehicles. We have identified altered supramolecular organisation and consequently distinct in vitro and in vivo performance of cell-targeted protein nanoparticles depending on material’s origin. It is a particularly important aspect regarding recombinant production of smart and complex protein structures Albeit it has been recently known that the bacterial host directly influence the architecture and the performance of the produced protein nanoparticles, how the genetic background of E. coli strains deficient of chaperons or LPS-free affect these protein assemblies has not been addressed. In this thesis we have investigated the fine structure of CXCR4 and CD44 targeted nanoparticles produced in different E. coli strains and how their physicochemical characteristics affect the nanoparticles’ biointeractions. Results illustrate the robustness of self-assembling patterns among tumor-targeted GFP nanoparticles and also the influence of genetic background of the producing cells on shifting the distribution of oligomeric population in the pooled material. The intrinsic variability derived from the bioproduction, although being unpredictable or uncontrollable, offers a scope of nanoparticulated material from where optimal population can be selected considering their ultimate application
10
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.
Books on the topic "Recombinant protein"
1
Tuan, Rocky S. Recombinant Protein Protocols. New Jersey: Humana Press, 1997. http://dx.doi.org/10.1385/089603481x.
Full text
2
Buckel, Peter, ed. Recombinant Protein Drugs. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7.
Full text
3
Bill, Roslyn M., ed. Recombinant Protein Production in Yeast. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-61779-770-5.
Full text
4
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 text
5
Jean, Garnier, ed. Introduction to proteins and protein engineering. Amsterdam: Elsevier, 1988.
Find full text
6
Jean, Garnier, ed. Introduction to proteins and protein engineering. Amsterdam: Elsevier, 1986.
Find full text
7
Hacker, David L., ed. Recombinant Protein Expression in Mammalian Cells. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-8730-6.
Full text
8
Howard, John A., and Elizabeth E. Hood, eds. Commercial Plant-Produced Recombinant Protein Products. Berlin, Heidelberg: Springer Berlin Heidelberg, 2014. http://dx.doi.org/10.1007/978-3-662-43836-7.
Full text
9
S, Tuan Rocky, ed. Recombinant protein protocols: Detection and isolation. Totowa, N.J: Humana Press, 1997.
Find full text
10
Robson, Barry. Introductionto proteins and protein engineering. Amsterdam: Elsevier, 1988.
Find full textBook chapters on the topic "Recombinant protein"
1
Fothergill-Gilmore, Linda A. "Recombinant Protein Technology." In Protein Biotechnology, 467–87. Totowa, NJ: Humana Press, 1993. http://dx.doi.org/10.1007/978-1-59259-438-2_13.
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
Rosenberg, Ian M. "Recombinant Protein Techniques." In Protein Analysis and Purification, 385–430. Boston, MA: Birkhäuser Boston, 1996. http://dx.doi.org/10.1007/978-1-4612-2056-5_11.
Full textConference papers on the topic "Recombinant protein"
1
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.
2
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.
3
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.
4
Qiu, Weiguo, Yiding Huang, Joseph Cappello, and Xiaoyi Wu. "Electrospun Recombinant Protein Polymer Nanofibers as a Biomaterial." In ASME 2010 First Global Congress on NanoEngineering for Medicine and Biology. ASMEDC, 2010. http://dx.doi.org/10.1115/nemb2010-13131.
Full textAbstract:
Micro/nanometer protein fibers are fundamental building blocks of extra-/intracellular matrices, providing structural support, stability and protection to cells, tissue and organism [1]. Fabricating performance protein micro/nanofibers has been extensively pursued for a variety of biomedical application. In this study, silk-elastinlike protein (SELP) polymer will be electrospun into nanofibers as a biomaterial.
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
Shetty, R. S., Lyndon L. Salins, S. Ramanathan, and Sylvia Daunert. "Recombinant methods in protein and whole-cell biosensing." In Photonics East '99, edited by Mahmoud Fallahi and Basil I. Swanson. SPIE, 1999. http://dx.doi.org/10.1117/12.372904.
Full text
7
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.
8
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.
9
Johnson, Daniel, Keat Teoh, Cody Ashby, Elizabeth Hood, and Xiuzhen Huang. "Analyzing genetic factors involved in recombinant protein expression enhancement." In 2010 IEEE International Conference on Bioinformatics and Biomedicine Workshops (BIBMW). IEEE, 2010. http://dx.doi.org/10.1109/bibmw.2010.5703806.
Full text
10
Pancham, N., M. Dumas, J. Brown, T. C. Michaud, and W. J. Knowles. "SYNTHETIC PEPTIDE ANTIBODIES RECOGNIZE PLASMA AND RECOMBINANT FVIII." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644027.
Full textAbstract:
Monoclonal antibodies were raised against synthetic peptides corresponding to the N-termini of the 90kd and 80kd subunits of human FVIII. Preliminary screening was performed directly against the peptides (linked to albumin) coated onto polystyrene wells. IgG was purified by Protein A-Sepharose and affinity purified using the immunogen peptides linked to Sepharose. Immunoreactivity with both plasma and recombinant FVIII was compared by Western blotting, two-site ELISA employing a neutralizing rabbit anti-FVIII IgG as capture antibody, and inhibition in a fluid phase FVIII activity assay. None of the antibodies neutralized clotting or amidolytic activities associated with either FVIII protein. All of the antibodies immunoblotted intacl or thrombin digested FVIII polypeptides according to their anticipated epitope recognition sites; this was useful in determining identity between the two types of FVIII protein. Furthermore, when either FVIII protein was bound to polyclonal IgG coated onto polystyrene microtiter wells, the 80kd antipeptide antibody was capable of detecting both antigens with equal affinity. These results, coupled with results using the 90k antipeptide suggest that epitope accessibility for these antipeptide antibodies is the same for plasma and recombinant FVIII under the conditions tested.
Reports on the topic "Recombinant protein"
1
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
2
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
3
Galili, Gad, and Alan Bennett. Role of Molecular Chaperone in Wheat Storage Protein Assembly. United States Department of Agriculture, April 1995. http://dx.doi.org/10.32747/1995.7604926.bard.
Full textAbstract:
Following sequestration into the ER, wheat gliadins assemble into complexes that initiate the formation of protein bodies. In the present work we have characterized the DNA sequence and regulation of expression of a plant BiP and also studied its interaction with wheat storage proteins as well as its role in the maturation of these storage proteins. In the Israeli lab, immunoprecipitation studies were made using anti BiP and anti storage proteins sera, both in wheat and in transgenic tobacco plants expressing a wheat gliadin storage proteins. In both cases, we could show that BiP interacts with the gliadin storage proteins. In addition, we could show that BiP also played an important role in the initial assembly of the gliadins. In the American lab, the complexity, structure and properties of tomato BiP was characterized at the molecular and biochemical levels. In addition, tomato BiP was also overexpressed in bacteria and the overexpressed protein was found to be active. The cooperative findings of the Israeli and American labs clearly improves our understanding of the structure and expression of a plant BiP as well as its role in the maturation of storage proteins in plants seeds. In addition, it will serve as a foundation for future studies of the mechanisms of BiP function in in vitro studies using purified storage proteins and purified recombinant active BiP.
4
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
5
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
6
Banai, Menachem, and Gary Splitter. Molecular Characterization and Function of Brucella Immunodominant Proteins. United States Department of Agriculture, July 1993. http://dx.doi.org/10.32747/1993.7568100.bard.
Full textAbstract:
The BARD project was a continuation of a previous BARD funded research project. It was aimed at characterization of the 12kDa immunodominant protein and subsequently the cloning and expression of the gene in E. coli. Additional immunodominant proteins were sought among genomic B. abortus expression library clones using T-lymphocyte proliferation assay as a screening method. The 12kDa protein was identified as the L7/L12 ribosomal protein demonstrating in the first time the role a structural protein may play in the development of the host's immunity against the organism. The gene was cloned from B. abortus (USA) and B. melitensis (Israel) showing identity of the oligonucleotide sequence between the two species. Further subcloning allowed expression of the protein in E. coli. While the native protein was shown to have DTH antigenicity its recombinant analog lacked this activity. In contrast the two proteins elicited lymphocyte proliferation in experimental murine brucellosis. CD4+ cells of the Th1 subset predominantly responded to this protein demonstrating the development of protective immunity (g-IFN, and IL-2) in the host. Similar results were obtained with bovine Brucella primed lymphocytes. UvrA, GroE1 and GroEs were additional Brucella immunodominant proteins that demonstrated MHC class II antigenicity. The role cytotoxic cells are playing in the clearance of brucella cells was shown using knock out mice defective either in their CD4+ or CD8+ cells. CD4+ defective mice were able to clear brucella as fast as did normal mice. In contrast mice which were defective in their CD8+ cells could not clear the organisms effectively proving the importance of this subtype cell line in development of protective immunity. The understanding of the host's immune response and the expansion of the panel of Brucella immunodominant proteins opened new avenues in vaccine design. It is now feasible to selectively use immunodominant proteins either as subunit vaccine to fortify immunity of older animals or as diagnostic reagents for the serological survaillance.
7
Palmer, Guy H., Eugene Pipano, Terry F. McElwain, Varda Shkap, and Donald P. Knowles, Jr. Development of a Multivalent ISCOM Vaccine against Anaplasmosis. United States Department of Agriculture, July 1993. http://dx.doi.org/10.32747/1993.7568763.bard.
Full textAbstract:
Anaplasmosis is an arthropod+borne disease of cattle caused by the rickettsia Anaplasma marginale and an impediment to efficient production of healthy livestock in both Israel and the United States. Our research focuses on development of a recombinant membrane surface protein (MSP) immunogen to replace current vaccines derived from the blood of infected cattle. The risk of widespread transmission of both known and newly emergent pathogens has prevented licensure of live blood-based vaccines in the U.S. and is a major concern for their continued use in Israel. Briefly, we accomplished the following in our BARD supported research: i) characterization of the intramolecular and intermolecular relationships of the native Major Surface Proteins (MSP) in the outer membrane; ii) expression, purification, and epitope characterization of the recombinant MSP-2, MSP-3, MSP-4, and MSP-5 proteins required to construct the recombinant ISCOM; iii) demonstration that the outer membrane-Quil A induces CD4+ T lymphocytes specific for the outer membrane polypeptides; iv) identification of CD4+ T lymphocytes that recognize outer membrane polypeptide epitopes conserved among other wise antigenically distinct strains; v) determination that immunization with the outer membrane-Quil A construct does not affect the ability of ticks to acquire or transmit A. marginale; and vi) demonstration that the outer membrane-Quil A construct induces complete protection against rickettsemia upon homologous challenge and significant protection against challenge with antigenically distinct strains, including tick transmission. Importantly, the level of protection against homologous challenge in the MSP vaccinates was comparable to that induced by live blood-based vaccines and demonstrates that development of a new generation of vaccines is feasible.
8
Dolja, Valerian V., Amit Gal-On, and Victor Gaba. Suppression of Potyvirus Infection by a Closterovirus Protein. United States Department of Agriculture, March 2002. http://dx.doi.org/10.32747/2002.7580682.bard.
Full textAbstract:
The plant virus family Polyviridae is the largest and most destructive of all plant viruses. Despite the continuous effort to develop resistant plant varieties, there is a desperate need for novel approaches conferring wide-range potyvirus resistance. Based on experiments with the tobacco etch potyvirus (TEV)-derived gene expression vector, we suggested approach for screening of the candidate resistance genes. This approach relies on insertion of the genes into a virus vector and evaluation of the phenotypes of the resulting recombinant viruses. The genes which suppress infection by the recombinant virus are selected as candidates for engineering transgenic resistance. Our analysis of the TEV variants expressing proteins of the beet yellows closterovirus (BYV) revealed that one of those, the leader proteinase (L-Pro), strongly and specifically interfered with the hybrid TEV infection. Since closterovirus L-Pro is evolutionary related to potyviral helper component-proteinase (HC-Pro), we suggested that the L-Pro interfered with HC-Pro function via a trans-dominant inhibitory effect. Based on these findings, we proposed to test two major hypotheses. First, we suggested that L-Pro-mediated suppression of potyvirus infection is a general phenomenon effective against a range of potyviruses. The second hypothesis stated that the suppression effect can be reproduced in transgenic plants expressing L-Pro, and can be utilized for generation of resistance to potyviruses. In accord with these hypotheses, we developed two original objectives of our proposal: A) to determine the range of the closterovirus-derived suppression of potyviral infection, and B) to try and utilize the L-Pro-mediated suppression for the development of transgenic resistance to potyviruses. In the first phase of the project, we have developed all major tools and technologies required for successful completion of the proposed research. These included TEV and ZYMV vectors engineered to express several closteroviral L-Pro variants, and generation of the large collection of transgenic plants. To our satisfaction, characterization of the infection phenotypes exhibited by chimeric TEV and ZYMV variants confirmed our first hypothesis. For instance, similar to TEV-L- Pro(BYV) chimera, ZYMV-L-Pro(LIYV) chimera was debilitated in its systemic spread. In contrast, ZYMV-GUS chimera (positive control) was competent in establishing vigorous systemic infection. These and other results with chimeric viruses indicated that several closteroviral proteinases inhibit long-distance movement of the potyviruses upon co-expression in infected plants. In order to complete the second objective, we have generated ~90 tobacco lines transformed with closteroviral L-Pro variants, as well as ~100 lines transformed with BYV Hsp70-homolog (Hsp70h; a negative control). The presence and expression of the trans gene in each line was initially confirmed using RT-PCR and RNA preparations isolated from plants. However, since detection of the trans gene-specific RNA can not guarantee production of the corresponding protein, we have also generated L-Pro- and Hsp70h-specific antisera using corresponding synthetic peptides. These antisera allowed us to confirm that the transgenic plant lines produced detectable, although highly variable levels of the closterovirus antigens. In a final phase of the project, we tested susceptibility of the transgenic lines to TEV infection. To this end, we determined that the minimal dilution of the TEV inoculum that is still capable of infecting 100% of nontransgenic plants was 1:20, and used 10 plants per line (in total, ~2,000 plants). Unfortunately, none of the lines exhibited statistically significant reduction in susceptibility. Although discouraging, this outcome prompted us to expand our experimental plan and conduct additional experiments. Our aim was to test if closteroviral proteinases are capable of functioning in trans. We have developed agroinfection protocol for BYV, and tested if co- expression of the L-Pro is capable of rescuing corresponding null-mutant. The clear-cut, negative results of these experiments demonstrated that L-Pro acts only in cis, thus explaining the lack of resistance in our transgenic plants. We have also characterized a collection of the L-Pro alanine- scanning mutants and found direct genetic evidence of the requirement for L-Pro in virus systemic spread. To conclude, our research supported by BARD confirmed one but not another of our original hypotheses. Moreover, it provided an important insight into functional specialization of the viral proteinases and generated set of tools and data with which we will be able to address the molecular mechanisms by which these proteins provide a variety of critical functions during virus life cycle.
9
Bercovier, Herve, Raul Barletta, and Shlomo Sela. Characterization and Immunogenicity of Mycobacterium paratuberculosis Secreted and Cellular Proteins. United States Department of Agriculture, January 1996. http://dx.doi.org/10.32747/1996.7573078.bard.
Full textAbstract:
Our long-term goal is to develop an efficient acellular vaccine against paratuberculosis based on protein antigen(s). A prerequisite to achieve this goal is to analyze and characterize Mycobacterium paratuberculosis (Mpt) secreted and cellular proteins eliciting a protective immune response. In the context of this general objective, we proposed to identify, clone, produce, and characterize: the Mpt 85B antigen and other Mpt immunoreactive secreted proteins, the Mpt L7/L12 ribosomal protein and other immunoreactive cellular proteins, Mpt protein determinants involved in invasion of epithelial cells, and Mpt protein antigens specifically expressed in macrophages. Paratuberculosis is still a very serious problem in Israel and in the USA. In the USA, a recent survey evaluated that 21.6% of the dairy herd were infected with Mpt resulting in 200-250 million dollars in annual losses. Very little is known on the virulence factors and on protective antigens of Mpt. At present, the only means of controlling this disease are culling or vaccination. The current vaccines do not allow a clear differentiation between infected and vaccinated animals. Our long-term goal is to develop an efficient acellular paratuberculosis vaccine based on Mpt protein antigen(s) compatible with diagnostic tests. To achieve this goal it is necessary to analyze and characterize secreted and cellular proteins candidate for such a vaccine. Representative Mpt libraries (shuttle plasmid and phage) were constructed and used to study Mpt genes and gene products described below and will be made available to other research groups. In addition, two approaches were performed which did not yield the expected results. Mav or Mpt DNA genes that confer upon Msg or E. coli the ability to invade and/or survive within HEp-2 cells were not identified. Likewise, we were unable to characterize the 34-39 kDa induced secreted proteins induced by stress factors due to technical difficulties inherent to the complexity of the media needed to support substantial M. pt growth. We identified, isolated, sequenced five Mpt proteins and expressed four of them as recombinant proteins that allowed the study of their immunological properties in sensitized mice. The AphC protein, found to be up regulated by low iron environment, and the SOD protein are both involved in protecting mycobacteria against damage and killing by reactive oxygen (Sod) and nitrogen (AhpC) intermediates, the main bactericidal mechanisms of phagocytic cells. SOD and L7/L12 ribosomal proteins are structural proteins constitutively expressed. 85B and CFP20 are both secreted proteins. SOD, L7/L12, 85B and CFP20 were shown to induce a Th1 response in immunized mice whereas AphC was shown by others to have a similar activity. These proteins did not interfere with the DTH reaction of naturally infected cows. Cellular immunity provides protection in mycobacterial infections, therefore molecules inducing cellular immunity and preferentially a Th1 pathway will be the best candidate for the development of an acellular vaccine. The proteins characterized in this grant that induce a cell-mediated immunity and seem compatible with diagnostic tests, are good candidates for the construction of a future acellular vaccine.
10
Gafny, Ron, A. L. N. Rao, and Edna Tanne. Etiology of the Rugose Wood Disease of Grapevine and Molecular Study of the Associated Trichoviruses. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575269.bard.
Full textAbstract:
Rugose wood is a complex disease of grapevines, characterized by modification of the woody cylinder of affected vines. The control of rugose wood is based on the production of healthy propagation material. Detection of rugose wood in grapevines is difficult and expensive: budwood from tested plants is grafted onto sensitive Vitis indicators and the appearance of symptoms is monitored for 3 years. The etiology of rugose wood is complex and has not yet been elucidated. Several elongated clostero-like viruses are consistently found in affected vines; one of them, grapevine virus A (GVA), is closely associated with Kober stem grooving, a component of the rugose wood complex. GVA has a single-stranded RNA genome of 7349 nucleotides, excluding a polyA tail at the 3' terminus. The GVA genome includes five open reading frames (ORFs 1-5). ORF 4, which encodes for the coat protein of GVA, is the only ORF for which the function was determined experimentally. The original objectives of this research were: 1- To produce antisera to the structural and non-structural proteins of GVA and GVB and to use these antibodies to establish an effective detection method. 2- Develop full length infectious cDNA clones of GVA and GVB. 3- Study the roll of GVA and GVB in the etiology of the grapevine rugose wood disease. 4- Determine the function of Trichovirus (now called Vitivirus) encoded genes in the virus life cycle. Each of the ORFs 2, 3, 4 and 5 genes of GVA were cloned and expressed in E. coli and used to produce antisera. Both the CP (ORF 4) and the putative MP (ORF 3) were detected with their corresponding antisera in-GVA infected N. benthamiana and grapevine. The MP was first detected at an early stage of the infection, 6-12 h after inoculation, and the CP 2-3 days after inoculation. The MP could be detected in GVA-infected grapevines that tested negative for CP, both with CP antiserum and with a commercially available ELISA kit. Antisera to ORF 2 and 5 encoded proteins could react with the recombinant proteins but failed to detect both proteins in GVA infected plants. A full-length cDNA clone of grapevine virus A (GVA) was constructed downstream from the bacteriophage T7 RNA polymerase promoter. Capped in vitro transcribed RNA was infectious in N. benthamiana and N. clevelandii plants. Symptoms induced by the RNA transcripts or by the parental virus were indistinguishable. The infectivity of the in vitro-transcribed RNA was confirmed by serological detection of the virus coat and movement proteins and by observation of virions by electron microscopy. The full-length clone was modified to include a gus reporter gene and gus activity was detected in inoculated and systemic leaves of infected plants. Studies of GVA mutants suggests that the coat protein (ORF 4) is essential for cell to cell movement, the putative movement protein (ORF 3) indeed functions as a movement protein and that ORF 2 is not required for virus replication, cell to cell or systemic movement. Attempts to infect grapevines by in-vitro transcripts, by inoculation of cDNA construct in which the virus is derived by the CaMV 35S promoter or by approach grafting with infected N. benthamiana, have so far failed. Studies of the subcellular distribution of GFP fusion with each of ORF 2, 3 and 4 encoded protein showed that the CP fusion protein accumulated as a soluble cytoplasmatic protein. The ORF 2 fusion protein accumulated in cytoplasmatic aggregates. The MP-GFP fusion protein accumulated in a large number of small aggregates in the cytoplasm and could not move from cell to cell. However, in conditions that allowed movement of the fusion protein from cell to cell (expression by a PVX vector or in young immature leaves) the protein did not form cytoplasmatic aggregates but accumulated in the plasmodesmata.