Journal articles: 'ProteÃna recombinante'

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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López Domínguez, Jaime, Karla María Moran Sarmina, Denisse Placier Sosa, and Aracely López Monteon. "TECNOLOGÍA DEL ADN RECOMBINANTE." Kuxulkab 23, no. 47 (June 25, 2018): 41. http://dx.doi.org/10.19136/kuxulkab.a23n47.2627.

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La tecnología del ADN recombinante ha permitido la manipulación de la información genética de cualquier tipo de organismo, por lo que al día de hoy se han presentado grandes avances en cuanto a su desarrollo y aplicación en otras ramas, siendo una herramienta primordial en la creación de alternativas médicas para el control y prevención de enfermedades, tal es el caso de la obtención de la insulina recombinante humana que utiliza esta tecnología para tener un mayor rendimiento en la producción, así como para evitar las reacciones adversas que se presentaban con los productos obtenidos de origen animal. Por tal motivo, es importante comprender esta tecnología ya que con ello se han abierto las puertas a la producción de medicamentos y enzimas que pueden ayudar al tratamiento terapéutico de múltiples enfermedades. Palabras clave: Genes; clonación; proteína recombinante. Keywords: Genes; cloning; recombinant protein.

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Ingerslev, Jørgen, Kirsten Christiansen, Hanne Ravn, Edward Gomperts, and Gordon Bray. "Antibodies to Heterologous Proteins in Hemophilia A Patients Receiving Recombinant Factor VIII (Recombinate™)." Thrombosis and Haemostasis 87, no. 04 (2002): 626–34. http://dx.doi.org/10.1055/s-0037-1613059.

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SummaryAs a consequence of the manufacturing process, trace quantities of Chinese hamster ovary cell protein, bovine serum albumin and murine immunoglobulin G are present in Recombinate™ recombinant human factor VIII (rhFVIII). The development of antibodies (Abs) to these heterologous proteins was evaluated during long-term rhFVIII therapy of hemophilia A in 68 previously treated and 73 previously untreated patients. Ab prevalence was also assessed in 157 non-hemophilic subjects. Abs against heterologous proteins could be detected in varying percentages of patients and non-hemophilic subjects. Abs arose in patients sporadically, and levels were typically low. There were no adverse events associated with development or presence of anti-heterologous protein Abs. These data indicate that sustained immune responses to trace levels of heterologous proteins are very infrequent during long-term rhFVIII therapy.

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Mahadevappa, Mamatha, Richard A. DeScenzo, and Roger P. Wise. "Recombination of alleles conferring specific resistance to powdery mildew at the Mla locus in barley." Genome 37, no. 3 (June 1, 1994): 460–68. http://dx.doi.org/10.1139/g94-064.

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In barley (Hordeum vulgare L.), the Mla locus conditions reaction to the powdery mildew fungus Erysiphe graminis f.sp. hordei. Enrichment for genetic recombinants in the Mla region is possible by screening for recombination events between the flanking endosperm storage proteins hordeins C and B. Reciprocal crosses were made between the Franger (C.I. 16151) and Rupee (C.I. 16155) lines carrying the (Mla6 + Mla14) and Mla13 alleles, respectively. Recombinants were identified from F2 segregants by analyzing the extracted hordein polypeptides by sodium dodecyl sulphate – polyacrylamide gel electrophoresis. Two hundred and seventy-six recombinant gametes were identified from the 1800 seeds that were screened. Recombination of Mla alleles was analyzed by inoculating F4 recombinant lines with three isolates of E. graminis (A27, 5874, and CR3), which recognize specific Mla alleles. The linkage order established is Hor1–Mla6–Mla13–Mla14–Hor2. The genetic distances between Hor1–Mla6, Mla6–Mla13, and Mla13–Hor2, obtained using Mapmaker 3.0b F3 intercross analysis, are 3.9, 0.2, and 5.2 cM, respectively.Key words: recombinant, barley, powdery mildew, Mla, hordein.

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Rodrigues, P. R. C., R. C. Cunha, F. D. S. Santos, V. S. Gonçalves, P. M. M. Albuquerque, A. G. Santos Júnior, M. Lima, and F. P. L. Leite. "Expressão e caracterização da glicoproteína D do herpesvírus equídeo 1 em Pichia pastoris." Arquivo Brasileiro de Medicina Veterinária e Zootecnia 72, no. 3 (May 2020): 703–10. http://dx.doi.org/10.1590/1678-4162-11356.

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RESUMO O herpesvírus equídeo 1 (EHV-1) apresenta distribuição mundial e causa graves prejuízos à equideocultura. É agente de surtos de doença respiratória, reprodutiva e neurológica, em equídeos jovens e adultos. A glicoproteína D (gD) do envelope viral é essencial para ligação e penetração em células permissivas e direcionamento do sistema imunológico do hospedeiro, induz respostas imunes humorais e celulares, sendo um antígeno apropriado para ser utilizado em vacinas e imunodiagnóstico. O objetivo deste trabalho foi expressar e caracterizar a gD do EHV-1 em Pichia pastoris para posterior utilização como antígeno em técnicas de imunodiagnóstico e formulação de vacinas recombinantes. Uma sequência de DNA que codifica uma forma truncada da gDEHV-1 foi clonada no vetor pPICZαA de expressão em P. pastoris. Obteve-se uma proteína de ~41 kDa, como esperado. A proteína apresentou glicosilação entre 4 kDa e 16 kDa, demonstrada por deglicosilação enzimática. A proteína recombinante foi caracterizada antigenicamente e imunogenicamente por Western blot, utilizando-se anticorpos policlonais equinos anti-EHV-1, e por ELISA indireto em modelo murino, demonstrando que a gD recombinante manteve epítopos similares aos da proteína nativa. Esses resultados sugerem que a gDEHV-1 é um antígeno promissor para uso como imunobiológico no controle do EHV-1.

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Santos Junior, Alceu Gonçalves, Renan Eugênio Araujo Piraine, Rodrigo Casquero Cunha, Leandro Quintana Nizoli, Renato Andreotti, and Fábio Pereira Leivas Leite. "AVALIAÇÃO DE MÉTODOS PARA OBTENÇÃO DE PROTEÍNAS RECOMBINANTES." Science And Animal Health 5, no. 2 (November 6, 2017): 166. http://dx.doi.org/10.15210/sah.v5i2.11387.

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A utilização de Pichia pastoris para a produção de proteínas recombinantes apresenta inúmeras vantagens que esse sistema agrega, destacando-se a exportação das moléculas para o meio extracelular. Isto permite que a obtenção das proteínas possa ser realizada por métodos de precipitação, com uso de sais neutros ou solventes orgânicos, sem trazer prejuízos. O objetivo deste trabalho foi avaliar a utilização de sulfato de amônio, acetona e metanol para obtenção da proteína recombinante Bm86 do carrapato Rhipicephalus microplus expressa por P. pastoris. A ação provocada pelos solventes orgânicos ocasionou uma solubilidade parcial das proteínas. Na quantificação por densitometria da proteína rBm86-CG, verificou-se que utilizando acetona (1:2) foi obtido 30,3 mg/L, metanol (1:2) 60,5 mg/L e com sulfato de amônio a 70% (83,5 mg/L). A manutenção das características antigênicas após a precipitação foi verificada pela técnica de Western Blot, sendo que a proteína rBm86-CG obtida pelos três métodos foi reconhecida por anticorpos específicos. Os resultados sugerem que a precipitação com sais neutros e solventes orgânicos pode ser utilizada para obtenção de proteínas recombinantes como a rBm86-CG, sem trazer prejuízos a sua antigenicidade.

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Franke, C. A., C. M. Rice, J. H. Strauss, and D. E. Hruby. "Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants." Molecular and Cellular Biology 5, no. 8 (August 1985): 1918–24. http://dx.doi.org/10.1128/mcb.5.8.1918.

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The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.

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Franke, C. A., C. M. Rice, J. H. Strauss, and D. E. Hruby. "Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants." Molecular and Cellular Biology 5, no. 8 (August 1985): 1918–24. http://dx.doi.org/10.1128/mcb.5.8.1918-1924.1985.

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The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.

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Koval, Olga, Galina Kochneva, Anastasiya Tkachenko, Olga Troitskaya, Galina Sivolobova, Antonina Grazhdantseva, Anna Nushtaeva, Elena Kuligina, and Vladimir Richter. "Recombinant Vaccinia Viruses Coding Transgenes of Apoptosis-Inducing Proteins Enhance Apoptosis But Not Immunogenicity of Infected Tumor Cells." BioMed Research International 2017 (2017): 1–14. http://dx.doi.org/10.1155/2017/3620510.

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Genetic modifications of the oncolytic vaccinia virus (VV) improve selective tumor cell infection and death, as well as activation of antitumor immunity. We have engineered a double recombinant VV, coding human GM-CSF, and apoptosis-inducing protein apoptin (VV-GMCSF-Apo) for comparing with the earlier constructed double recombinant VV-GMCSF-Lact, coding another apoptosis-inducing protein, lactaptin, which activated different cell death pathways than apoptin. We showed that both these recombinant VVs more considerably activated a set of critical apoptosis markers in infected cells than the recombinant VV coding GM-CSF alone (VV-GMCSF-dGF): these were phosphatidylserine externalization, caspase-3 and caspase-7 activation, DNA fragmentation, and upregulation of proapoptotic protein BAX. However, only VV-GMCSF-Lact efficiently decreased the mitochondrial membrane potential of infected cancer cells. Investigating immunogenic cell death markers in cancer cells infected with recombinant VVs, we demonstrated that all tested recombinant VVs were efficient in calreticulin and HSP70 externalization, decrease of cellular HMGB1, and ATP secretion. The comparison of antitumor activity against advanced MDA-MB-231 tumor revealed that both recombinants VV-GMCSF-Lact and VV-GMCSF-Apo efficiently delay tumor growth. Our results demonstrate that the composition of GM-CSF and apoptosis-inducing proteins in the VV genome is very efficient tool for specific killing of cancer cells and for activation of antitumor immunity.

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Higgins, S. J., P. Young, J. R. Brody, and G. R. Cunha. "Induction of functional cytodifferentiation in the epithelium of tissue recombinants. I. Homotypic seminal vesicle recombinants." Development 106, no. 2 (June 1, 1989): 219–34. http://dx.doi.org/10.1242/dev.106.2.219.

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Functional cytodifferentiation of seminal vesicle epithelium was investigated in tissue recombinants. Neonatal rat and mouse seminal vesicles were separated into epithelium and mesenchyme using trypsin. Epithelium and mesenchyme were then recombined in vitro to form interspecific rat/mouse homotypic recombinants. Growth as renal grafts in adult male athymic mice resulted in seminal vesicle morphogenesis in 70% of the recombinants (the remaining 30% failed to grow). Functional cytodifferentiation was judged by the expression of the major androgen-dependent secretory proteins characteristic of the seminal vesicles of adult rats and mice. Antibodies specific for each of these proteins were used to screen tissue sections by immunocytochemistry and to probe protein extracts by immunoblotting techniques. The heterospecific recombinants synthesized the full range of seminal vesicle secretory proteins that typifies the species providing the epithelium of the recombinant, not the mesenchyme. There was little functional variation between individual recombinants. The time course of development corresponded to that of intact neonatal seminal vesicles grown under the same conditions. Morphogenesis and functional cytodifferentiation were not evident after one week, but were well advanced after two weeks. Seminal vesicle recombinants grown for three weeks were indistinguishable morphologically and functionally from normal adult seminal vesicles. In addition, the ability of adult seminal vesicle epithelium to be induced to proliferate was examined. In association with neonatal seminal vesicle mesenchyme, the epithelium of the adult seminal vesicle proliferated and retained its normal functional activity. Thus, seminal vesicle functional cytodifferentiation can be faithfully reproduced in homotypic tissue recombinants. The methods used in this study will be used to investigate seminal vesicle development in instructive inductions of heterotypic epithelia.

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Burnouf, T. "Recombinant plasma proteins." Vox Sanguinis 100, no. 1 (December 22, 2010): 68–83. http://dx.doi.org/10.1111/j.1423-0410.2010.01384.x.

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Geisow, Michael J. "Characterizing Recombinant Proteins." Bio/Technology 9, no. 10 (October 1991): 921–24. http://dx.doi.org/10.1038/nbt1091-921.

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Blanco, Rafael D., Cintia F. Fidelis, Leandro S. Araujo, Adriana M. Henao, Jose A. Cardona, Jose D. Guimarães, Marlene I. Vargas, and Joaquin H. Patarroyo. "Desenvolvimento e padronização do Dot-ELISA usando peptídeos recombinantes para o diagnóstico sorológico de Neospora caninum." Pesquisa Veterinária Brasileira 34, no. 8 (August 2014): 723–27. http://dx.doi.org/10.1590/s0100-736x2014000800002.

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A neosporose é reconhecida como uma das maiores causas de aborto e perdas neonatais em bovinos de leite e corte em todo o mundo. Nos últimos anos esta doença tem atraído o interesse de pesquisadores com foco na epidemiologia e métodos eficazes de diagnóstico desta doença. No presente estudo objetivou-se desenvolver e padronizar um teste Dot-ELISA para o diagnóstico sorológico de Neospora caninum com um peptídeo recombinate como antígeno, visando o desenvolvimento de um kit para diagnóstico a campo. O peptídeo recombinante (rNcGRA1) foi desenhado com base na metodologia de genética reversa de epítopos antigênicos originados de uma proteína de grânulos densos de N. caninum, e sintetizado pela GenScript (USA). Produzido mediante o processo fermentativo em leveduras Pichia pastoris KM71. Para a padronização do Dot-ELISA, membranas de nitrocelulose de 0.22µm foram sensibilizadas com 1µL do antígeno e posteriormente os soros foram diluídos em solução de lavagem e incubados durante 1 hora. A revelação foi feita mediante a adição de Proteína G marcada com peroxidase por 30 minutos, seguido da solução reveladora a base de 3,3’-Diaminobenzidine (DAB). Logo após a padronização foram testados 44 soros bovinos diagnosticados por imunofluorescência indireta (RIFI), obtendo-se uma concordância nos resultados do teste de 95,5% e uma sensibilidade e especificidade de 100% e 92% respectivamente. Quanto ao Kit para diagnóstico a campo na Plataforma Tecnológica RapidFlow-Through Miriad®, o peptídeo rNcGRA1 apresentou marcações visíveis ao reagir com os soros positivos, e não apresentou marcações usando os soros negativos. Este estudo é o primeiro a utilizar peptídeos recombinantes e mostrar-se eficiente para o diagnóstico sorológico de bovinos naturalmente infetados por N. caninum.

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Pham, Vu Minh, Huy Hua Hoang Quoc, Huong-Xuan Mai Le, and Tri-Nhan Nguyen*. "Storage of the Recombinant Protein hPDGF-BB in the Culture of Pichia pastoris." International Journal of Life-Sciences Scientific Research 4, no. 4 (July 2018): 1934–39. http://dx.doi.org/10.21276/ijlssr.2018.4.4.11.

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24

Buonocore, Linda, Keril J. Blight, Charles M. Rice, and John K. Rose. "Characterization of Vesicular Stomatitis Virus Recombinants That Express and Incorporate High Levels of Hepatitis C Virus Glycoproteins." Journal of Virology 76, no. 14 (July 15, 2002): 6865–72. http://dx.doi.org/10.1128/jvi.76.14.6865-6872.2002.

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ABSTRACT We generated recombinant vesicular stomatitis viruses (VSV) expressing genes encoding hybrid proteins consisting of the extracellular domains of hepatitis C virus (HCV) glycoproteins fused at different positions to the transmembrane and cytoplasmic domains of the VSV G glycoprotein (E1G and E2G). We show that these chimeric proteins are transported to the cell surface and incorporated into VSV virions efficiently. We also generated VSV recombinants in which the gene encoding the VSV G protein was deleted and replaced by one or both of the E1G and E2G genes, together with a green fluorescent protein gene. These ΔG viruses incorporated E1G and E2G proteins at levels approximately equivalent to the normal level of VSV G itself, or about 1,200 molecules of each protein per virion. Given the potency of VSV recombinants as vaccines in other studies, this high-level expression and incorporation of HCV proteins into virions could be very important for development of an HCV vaccine. Despite the presence of E1G and E2G proteins at high levels in the virions, these virions did not infect cell lines that have been reported to support at least a low level of HCV infection and replication.

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Abouelasrar Salama, Sara, Mirre De Bondt, Nele Berghmans, Mieke Gouwy, Vivian Louise Soares de Oliveira, Sergio C. Oliveira, Flavio A. Amaral, et al. "Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case." Mediators of Inflammation 2020 (July 6, 2020): 1–17. http://dx.doi.org/10.1155/2020/6087109.

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The serum amyloid A (SAA) gene family is highly conserved and encodes acute phase proteins that are upregulated in response to inflammatory triggers. Over the years, a considerable amount of literature has been published attributing a wide range of biological effects to SAAs such as leukocyte recruitment, cytokine and chemokine expression and induction of matrix metalloproteinases. Furthermore, SAAs have also been linked to protumorigenic, proatherogenic and anti-inflammatory effects. Here, we investigated the biological effects conveyed by murine SAA3 (mu rSAA3) recombinantly expressed in Escherichia coli. We observed the upregulation of a number of chemokines including CCL2, CCL3, CXCL1, CXCL2, CXCL6 or CXCL8 following stimulation of monocytic, fibroblastoid and peritoneal cells with mu rSAA3. Furthermore, this SAA variant displayed potent in vivo recruitment of neutrophils through the activation of TLR4. However, a major problem associated with proteins derived from recombinant expression in bacteria is potential contamination with various bacterial products, such as lipopolysaccharide, lipoproteins and formylated peptides. This is of particular relevance in the case of SAA as there currently exists a discrepancy in biological activity between SAA derived from recombinant expression and that of an endogenous source, i.e. inflammatory plasma. Therefore, we subjected commercial recombinant mu rSAA3 to purification to homogeneity via reversed-phase high-performance liquid chromatography (RP-HPLC) and re-assessed its biological potential. RP-HPLC-purified mu rSAA3 did not induce chemokines and lacked in vivo neutrophil chemotactic activity, but retained the capacity to synergize with CXCL8 in the activation of neutrophils. In conclusion, experimental results obtained when using proteins recombinantly expressed in bacteria should always be interpreted with care.

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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.

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Gunneriusson, Elin, Patrik Samuelson, Jenny Ringdahl, Hans Grönlund, Per-Åke Nygren, and Stefan Ståhl. "Staphylococcal Surface Display of Immunoglobulin A (IgA)- and IgE-Specific In Vitro-Selected Binding Proteins (Affibodies) Based on Staphylococcus aureus Protein A." Applied and Environmental Microbiology 65, no. 9 (September 1, 1999): 4134–40. http://dx.doi.org/10.1128/aem.65.9.4134-4140.1999.

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ABSTRACT An expression system designed for cell surface display of hybrid proteins on Staphylococcus carnosus has been evaluated for the display of Staphylococcus aureus protein A (SpA) domains, normally binding to immunoglobulin G (IgG) Fc but here engineered by combinatorial protein chemistry to yield SpA domains, denoted affibodies, with new binding specificities. Such affibodies, with human IgA or IgE binding activity, have previously been selected from a phage library, based on an SpA domain. In this study, these affibodies have been genetically introduced in monomeric or dimeric forms into chimeric proteins expressed on the surface of S. carnosus by using translocation signals from aStaphylococcus hyicus lipase construct together with surface-anchoring regions of SpA. The recombinant surface proteins, containing the IgA- or IgE-specific affibodies, were demonstrated to be expressed as full-length proteins, localized and properly exposed at the cell surface of S. carnosus. Furthermore, these chimeric receptors were found to be functional, since recombinantS. carnosus cells were shown to have gained IgA and IgE binding capacity, respectively. In addition, a positive effect in terms of IgA and IgE reactivity was observed when dimeric versions of the affibodies were present. Potential applications for recombinant bacteria with redirected binding specificity in their surface proteins are discussed.

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BRUCHHAUS, Iris, Symi RICHTER, and Egbert TANNICH. "Removal of hydrogen peroxide by the 29 kDa protein of Entamoeba histolytica." Biochemical Journal 326, no. 3 (September 15, 1997): 785–89. http://dx.doi.org/10.1042/bj3260785.

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The 29 kDa protein of Entamoeba histolytica (Eh29), as well as a truncated variant of this protein, which lacks a cysteine-rich N-terminal region of 40 amino acid residues (Eh29mut), were recombinantly expressed in Escherichia coli and purified to homogeneity. Both recombinant proteins (recEh29, recEh29mut) were found to have hydrogen peroxide (H2O2)-removing activity, but recEh29 was twice as active as recEh29mut. For the consumption of exogenous H2O2, activity was dependent on the presence of reducing equivalents, such as dithiothreitol (DTT), indicating that Eh29 constitutes a thiol-dependent peroxidase. DTT was not required to remove H2O2 by recEh29 or recEh29mut when H2O2 was generated enzymically by the E. histolytica NADPH:flavin oxidoreductase. This enzyme produces H2O2 under aerobic conditions and simultaneously serves as a hydrogen donor for Eh29. Peroxidase activity of the recombinant proteins was further supported by complementation of an E. coli strain that lacks the entire alkyl hydroperoxide reductase locus. The high sensitivity of these bacteria against cumene hydroperoxide was significantly reduced by the introduction of the genes encoding recEh29 or recEh29mut. Using antisera raised against the recombinant proteins, native Eh29 was localized within the cytoplasm of the amoebae. In addition, the antisera reacted with proteins of E. histolytica lysates with apparent molecular masses of 35 kDa and 160–300 kDa. All of them exhibited thiol-peroxidase activity.

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Wingfield, Paul T., Robert J. Mattaliano, H. Robson MacDonald, Stewart Craig, G. Marius Clore, Angela M. Gronenborn, and Ursula Schmeissner. "Recombinant-derived interleukin-1α stabilized against specific deamidation." "Protein Engineering, Design and Selection" 1, no. 5 (1987): 413–17. http://dx.doi.org/10.1093/protein/1.5.413.

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Traktman, Paula, Ke Liu, Joseph DeMasi, Robert Rollins, Sophy Jesty, and Beth Unger. "Elucidating the Essential Role of the A14 Phosphoprotein in Vaccinia Virus Morphogenesis: Construction and Characterization of a Tetracycline-Inducible Recombinant." Journal of Virology 74, no. 8 (April 15, 2000): 3682–95. http://dx.doi.org/10.1128/jvi.74.8.3682-3695.2000.

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ABSTRACT We have previously reported the construction and characterization of vindH1, an inducible recombinant in which expression of the vaccinia virus H1 phosphatase is regulated experimentally by IPTG (isopropyl-β-d-thiogalactopyranoside) (35). In the absence of H1 expression, the transcriptional competence and infectivity of nascent virions are severely compromised. We have sought to identify H1 substrates by characterizing proteins that are hyperphosphorylated in H1-deficient virions. Here, we demonstrate that the A14 protein, a component of the virion membrane, is indeed an H1 phosphatase substrate in vivo and in vitro. A14 is hyperphosphorylated on serine residues in the absence of H1 expression. To enable a genetic analysis of A14's function during the viral life cycle, we have adopted the regulatory components of the tetracycline (TET) operon and created new reagents for the construction of TET-inducible vaccinia virus recombinants. In the context of a virus expressing the TET repressor (tetR), insertion of the TET operator between the transcriptional and translational start sites of a late viral gene enables its expression to be tightly regulated by TET. We constructed a TET-inducible recombinant for the A14 gene, vindA14. In the absence of TET, vindA14 fails to form plaques and the 24-h yield of infectious progeny is reduced by 3 orders of magnitude. The infection arrests early during viral morphogenesis, with the accumulation of large numbers of vesicles and the appearance of “empty” crescents that appear to adhere only loosely to virosomes. This phenotype corresponds closely to that observed for an IPTG-inducible A14 recombinant whose construction and characterization were reported while our work was ongoing (47). The consistency in the phenotypes seen for the IPTG- and TET-inducible recombinants confirms the efficacy of the TET-inducible system and reinforces the value of having a second, independent system available for generating inducible recombinants.

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Fedorov, T. V., T. V. Reshetnyak, E. A. Panfertsev, and S. F. Biketov. "Renaturation of recombinant proteins." Bacteriology 4, no. 4 (2019): 19–28. http://dx.doi.org/10.20953/2500-1027-2019-4-19-28.

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32

Buckel, Peter. "Recombinant proteins for therapy." Trends in Pharmacological Sciences 17, no. 12 (December 1996): 450–56. http://dx.doi.org/10.1016/s0165-6147(96)01011-5.

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De Bernardez Clark, Eliana. "Refolding of recombinant proteins." Current Opinion in Biotechnology 9, no. 2 (April 1998): 157–63. http://dx.doi.org/10.1016/s0958-1669(98)80109-2.

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Bialy, Harvey. "Recombinant Proteins: Virtual Authenticity." Nature Biotechnology 5, no. 9 (September 1987): 883–90. http://dx.doi.org/10.1038/nbt0987-883.

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35

Tullius, Michael V., Günter Harth, and Marcus A. Horwitz. "High Extracellular Levels of Mycobacterium tuberculosis Glutamine Synthetase and Superoxide Dismutase in Actively Growing Cultures Are Due to High Expression and Extracellular Stability Rather than to a Protein-Specific Export Mechanism." Infection and Immunity 69, no. 10 (October 1, 2001): 6348–63. http://dx.doi.org/10.1128/iai.69.10.6348-6363.2001.

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ABSTRACT Glutamine synthetase (GS) and superoxide dismutase (SOD), large multimeric enzymes that are thought to play important roles in the pathogenicity of Mycobacterium tuberculosis, are among the bacterium's major culture filtrate proteins in actively growing cultures. Although these proteins lack a leader peptide, their presence in the extracellular medium during early stages of growth suggested that they might be actively secreted. To understand their mechanism of export, we cloned the homologous genes (glnA1 andsodA) from the rapid-growing, nonpathogenicMycobacterium smegmatis, generated glnA1 andsodA mutants of M. smegmatis by allelic exchange, and quantitated expression and export of both mycobacterial and nonmycobacterial GSs and SODs in these mutants. We also quantitated expression and export of homologous and heterologous SODs fromM. tuberculosis. When each of the genes was expressed from a multicopy plasmid, M. smegmatis exported comparable proportions of both the M. tuberculosis andM. smegmatis GSs (in the glnA1 strain) or SODs (in the sodA strain), in contrast to previous observations in wild-type strains. Surprisingly, recombinantM. smegmatis and M. tuberculosisstrains even exported nonmycobacterial SODs. To determine the extent to which export of these large, leaderless proteins is expression dependent, we constructed a recombinant M. tuberculosis strain expressing green fluorescent protein (GFP) at high levels and a recombinant M. smegmatis strain coexpressing the M. smegmatis GS, M. smegmatis SOD, and M. tuberculosis BfrB (bacterioferritin) at high levels. The recombinant M. tuberculosis strain exported GFP even in early stages of growth and at proportions very similar to those of the endogenousM. tuberculosis GS and SOD. Similarly, the recombinantM. smegmatis strain exported bacterioferritin, a large (∼500-kDa), leaderless, multimeric protein, in proportions comparable to GS and SOD. In contrast, high-level expression of the large, leaderless, multimeric protein malate dehydrogenase did not lead to extracellular accumulation because the protein was highly unstable extracellularly. These findings indicate that, contrary to expectations, export of M. tuberculosis GS and SOD in actively growing cultures is not due to a protein-specific export mechanism, but rather to bacterial leakage or autolysis, and that the extracellular abundance of these enzymes is simply due to their high level of expression and extracellular stability. The same determinants likely explain the presence of other leaderless proteins in the extracellular medium of actively growing M. tuberculosis cultures.

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Gabibov, A. G. "Recombinant antibodies and recombinant proteins: Prolonged-action drugs." Herald of the Russian Academy of Sciences 86, no. 3 (May 2016): 169–73. http://dx.doi.org/10.1134/s1019331616030114.

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Nova-López, Carlos Julio, Jorge Mario Muñoz-Pérez, Luisa Fernanda Granger-Serrano, Mario Eveilio Arias-Zabala, and Rafael Eduardo Arango-Isaza. "Expresión de la proteína recombinante Cry 1Ac en cultivos de células de papa en suspensión: Establecimiento del cultivo y optimización de la producción de la biomasa y la proteína mediante la adición de nitrógeno." DYNA 84, no. 201 (April 1, 2017): 34. http://dx.doi.org/10.15446/dyna.v84n201.59829.

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Los cultivos in vitro de células vegetales en suspensión se han propuesto como plataformas alternativas de expresión de proteínas recombinantes con aplicación terapéutica por las ventajas que ofrecen sobre los sistemas tradicionales de expresión en células bacterianas y de mamíferos. En este trabajo se determinó un protocolo para el establecimiento de suspensiones de papa (S. tuberosum) genéticamente modificadas con el gen de la proteína Cry 1Ac y se caracterizaron las cinéticas de producción de la biomasa y la proteína recombinante. Los entrenudos y el medio MS suplementado con 2.0 mg L-1 de 2,4-D, mostraron los mejores porcentajes de formación de callo. La tasa máxima de crecimiento específico calculada para las suspensiones fue 0.12 d-1, con una concentración máxima de biomasa de 1.41 g L-1 al final de la fase exponencial, la cual logró aumentarse hasta 3.94 g L-1 duplicando la concentración de NO3- y NH4+ en el medio de cultivo.

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Schönherr, Robert, Janine Mia Rudolph, and Lars Redecke. "Protein crystallization in living cells." Biological Chemistry 399, no. 7 (June 27, 2018): 751–72. http://dx.doi.org/10.1515/hsz-2018-0158.

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AbstractProtein crystallization in living cells has been observed surprisingly often as a native assembly process during the past decades, and emerging evidence indicates that this phenomenon is also accessible for recombinant proteins. But only recently the advent of high-brilliance synchrotron sources, X-ray free-electron lasers, and improved serial data collection strategies has allowed the use of these micrometer-sized crystals for structural biology. Thus,in cellulocrystallization could offer exciting new possibilities for proteins that do not crystallize applying conventional approaches. In this review, we comprehensively summarize the current knowledge of intracellular protein crystallization. This includes an overview of the cellular functions, the physical properties, and, if known, the mode of regulation of nativein cellulocrystal formation, complemented with a discussion of the reported crystallization events of recombinant proteins and the current method developments to successfully collect X-ray diffraction data fromin cellulocrystals. Although the intracellular protein self-assembly mechanisms are still poorly understood, regulatory differences between nativein cellulocrystallization linked to a specific function and accidently crystallizing proteins, either disease associated or recombinantly introduced, become evident. These insights are important to systematically exploit living cells as protein crystallization chambers in the future.

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Osman, Fekret, Irina R. Tsaneva, Matthew C. Whitby, and Claudette L. Doe. "UV Irradiation Causes the Loss of Viable Mitotic Recombinants in Schizosaccharomyces pombe Cells Lacking the G2/M DNA Damage Checkpoint." Genetics 160, no. 3 (March 1, 2002): 891–908. http://dx.doi.org/10.1093/genetics/160.3.891.

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Abstract Elevated mitotic recombination and cell cycle delays are two of the cellular responses to UV-induced DNA damage. Cell cycle delays in response to DNA damage are mediated via checkpoint proteins. Two distinct DNA damage checkpoints have been characterized in Schizosaccharomyces pombe: an intra-S-phase checkpoint slows replication and a G2/M checkpoint stops cells passing from G2 into mitosis. In this study we have sought to determine whether UV damage-induced mitotic intrachromosomal recombination relies on damage-induced cell cycle delays. The spontaneous and UV-induced recombination phenotypes were determined for checkpoint mutants lacking the intra-S and/or the G2/M checkpoint. Spontaneous mitotic recombinants are thought to arise due to endogenous DNA damage and/or intrinsic stalling of replication forks. Cells lacking only the intra-S checkpoint exhibited no UV-induced increase in the frequency of recombinants above spontaneous levels. Mutants lacking the G2/M checkpoint exhibited a novel phenotype; following UV irradiation the recombinant frequency fell below the frequency of spontaneous recombinants. This implies that, as well as UV-induced recombinants, spontaneous recombinants are also lost in G2/M mutants after UV irradiation. Therefore, as well as lack of time for DNA repair, loss of spontaneous and damage-induced recombinants also contributes to cell death in UV-irradiated G2/M checkpoint mutants.

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Kahn, Jeffrey S., Anjeanette Roberts, Carla Weibel, Linda Buonocore, and John K. Rose. "Replication-Competent or Attenuated, Nonpropagating Vesicular Stomatitis Viruses Expressing Respiratory Syncytial Virus (RSV) Antigens Protect Mice against RSV Challenge." Journal of Virology 75, no. 22 (November 15, 2001): 11079–87. http://dx.doi.org/10.1128/jvi.75.22.11079-11087.2001.

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ABSTRACT Foreign glycoproteins expressed in recombinant vesicular stomatitis virus (VSV) can elicit specific and protective immunity in the mouse model. We have previously demonstrated the expression of respiratory syncytial virus (RSV) G (attachment) and F (fusion) glycoprotein genes in recombinant VSV. In this study, we demonstrate the expression of RSV F and G glycoproteins in attenuated, nonpropagating VSVs which lack the VSV G gene (VSVΔG) and the incorporation of these RSV proteins into recombinant virions. We also show that intranasal vaccination of mice with nondefective VSV recombinants expressing RSV G (VSV-RSV G) or RSV F (VSV-RSV F) elicited RSV-specific antibodies in serum (by enzyme-linked immunosorbent assay [ELISA]) as well as neutralizing antibodies to RSV and afford complete protection against RSV challenge. In contrast, VSVΔG-RSV F induced detectable serum antibodies to RSV by ELISA, but no detectable neutralizing antibodies, yet it still protected from RSV challenge. VSVΔG-RSV G failed to induce any detectable serum (by ELISA) or neutralizing antibodies and failed to protect from RSV challenge. The attenuated, nonpropagating VSVΔG-RSV F is a particularly attractive candidate for a live attenuated recombinant RSV vaccine.

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Mei, Liu, Lu Lin-Jing, Huang Jun-Yan, Zhang Shu-Huan, Bi Ding-Ren, and Sun Ming. "Display of H5N1Avian influenza virushaemagglutinin HA1 onBacillus thuringiensiscell surface and its immunogenicity for mice." Chinese Journal of Agricultural Biotechnology 4, no. 3 (December 2007): 221–28. http://dx.doi.org/10.1017/s1479236207001702.

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AbstractThe S-layer protein CTC surface display system ofBacillus thuringiensiswas used to test the possibility of displaying H5N1Avian influenza virus(AIV) haemagglutinin HA1 on the cell surface ofB. thuringiensis. Two recombinant plasmids, pCTC-HA1P and pCSHA1P, were constructed by replacing the central part below the surface anchor sequenceslhof S-layer protein genectcwith partha1gene(ha1p). pCTC-HA1P harboured the fusion genectc-ha1pand pCSHA1P the fusion genecsa-ctc-ha1p,csarepresenting thecsaABoperon (very important in anchoring S-layer protein on the bacterial cell surface). Two recombinantB. thuringiensisstrains were constructed by electrotransferring recombinant plasmids toB. thuringiensisplasmid-free derivative strain BMB171. Strains obtained were CH (bearing pCSHA1P) and BCCH (bearing pCTC-HA1P as well as thecsaABoperon-carrying plasmid pMIL-CSA). The vegetative cells of CH and BCCH were used as antigens in haemagglutination (HA) and haemagglutination inhibition (HI) assays. HA assay showed recombinant HA1 proteins successfully displayed on the cell surface of CH and BCCH. HI assay showed that these recombinant HA1 proteins were specific to standard positive HI (haemagglutination inhibition test) serum of subtype H5 AIV. After immunization of mice with vegetative cells, both CH and BCCH elicited a humoral response to HA1 and exhibited immunogenicity as indicated by enzyme-linked immunosorbent assay (ELISA). ELISA also showed that CH exhibited a higher immunogenicity than BCCH. The strategy developed in this study suggests the possibility of generating a heat-stable and oral veterinary vaccine against AIV with theB. thuringiensisS-layer protein CTC surface display system.

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Berg, Michael G., Robert J. Adams, Ratish Gambhira, Mark C. Siracusa, Alan L. Scott, Richard B. S. Roden, and Gary Ketner. "Immune Responses in Macaques to a Prototype Recombinant Adenovirus Live Oral Human Papillomavirus 16 Vaccine." Clinical and Vaccine Immunology 21, no. 9 (July 2, 2014): 1224–31. http://dx.doi.org/10.1128/cvi.00197-14.

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ABSTRACTImmunization with human papillomavirus (HPV) L1 virus-like particles (VLPs) prevents infection with HPV. However, the expense and logistical demands of current VLP vaccines will limit their widespread use in resource-limited settings, where most HPV-induced cervical cancer occurs. Live oral adenovirus vaccines have properties that are well-suited for use in such settings. We have described a live recombinant adenovirus vaccine prototype that produces abundant HPV16 L1 protein from the adenovirus major late transcriptional unit and directs the assembly of HPV16 VLPs in tissue culture. Recombinant-derived VLPs potently elicit neutralizing antibodies in mice. Here, we characterize the immune response to the recombinant after dual oral and intranasal immunization of pigtail macaques, in which the virus replicates as it would in immunized humans. The immunization of macaques induced vigorous humoral responses to adenovirus capsid and nonstructural proteins, although, surprisingly, not against HPV L1. In contrast, immunization elicited strong T-cell responses to HPV VLPs as well as adenovirus virions. T-cell responses arose immediately after the primary immunization and were boosted by a second immunization with recombinant virus. T-cell immunity contributes to protection against a wide variety of pathogens, including many viruses. The induction of a strong cellular response by the recombinant indicates that live adenovirus recombinants have potential as vaccines for those agents. These studies encourage and will inform the continued development of viable recombinant adenovirus vaccines.

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Melo, Elaine S. P., Flábio R. Araújo, Carlos A. N. Ramos, Cleber O. Soares, Grácia M. S. Rosinha, Carina Elisei, and Cláudio R. Madruga. "ELISA com MSP5 recombinante truncada para detecção de anticorpos contra Anaplasma marginale em bovinos." Pesquisa Veterinária Brasileira 27, no. 7 (July 2007): 301–6. http://dx.doi.org/10.1590/s0100-736x2007000700008.

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Os objetivos deste estudo foram produzir e solubilizar a proteína MSP5 recombinante truncada de Anaplasma marginale, e avaliar seu desempenho em um ensaio de imunoadsorção enzimática indireto (ELISA) para detecção de anticorpos contra a riquétsia. O gene msp5, exceto a região N-terminal hidrofóbica, foi amplificado por PCR, clonado em plasmídeo pTrcHis-TOPO e expresso em Escherichia coli. A solubilização da proteína recombinante foi avaliada em diferentes pHs e concentrações de uréia. A sensibilidade e a especificidade do ensaio foram avaliados testando-se 66 soros de animais infectados experimentalmente com A. marginale e 96 soros negativos, com o estado de infecção destes animais confirmado por PCR. Um total de 1.666 amostras de soros bovino, provenientes do Brasil - Rio Grande do Sul (73), Mato Grosso do Sul (91), Pernambuco (86), Bahia (314) e Minas Gerais (267)-, Uruguai (32) e Costa Rica (803) foram testadas nos ELISAs com MSP5 truncada e com MSP1a recombinantes e a concordância entre os dois testes foi avaliada. O ELISA indireto com MSP5 truncada foi capaz de detectar animais infectados com 96,97% de sensibilidade e 100% de especificidade. Nos animais infectados experimentalmente, o ELISA detectou anticorpos do 12º até o último dia de observação (37º dia). Os ELISAs para MSP5 e MSP1a apresentaram concordância de 95,67%, com índice kappa de 0,81. Os resultados discordantes apresentaram uma diferença significativa (p <0,001). Anticorpos contra A. marginale foram detectados em animais de todas as regiões estudadas. O ELISA com MSP5 recombinante truncada apresentou bom desempenho na detecção de anticorpos contra A. marginale, com alta sensibilidade e especificidade, representando uma importante ferramenta para o diagnóstico da anaplasmose bovina em estudos epidemiológicos.

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Isore, D. P., T. K. Goswami, P. Chaudhury, S. K. Mukhopadhayay, and S. Ganguly. "Recombinant Protein and DNA Vaccine Construct of Brucella abortus L7/L12 Gene Elicits Immune Response." Asian Pacific Journal of Health Sciences 1, no. 4 (October 2014): 425–37. http://dx.doi.org/10.21276/apjhs.2014.1.4.20.

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Wood, Andrew, Benjamin L. J. Webb, Birke Bartosch, Torsten Schaller, Yasuhiro Takeuchi, and Greg J. Towers. "Porcine endogenous retroviruses PERV A and A/C recombinant are insensitive to a range of divergent mammalian TRIM5α proteins including human TRIM5α." Journal of General Virology 90, no. 3 (March 1, 2009): 702–9. http://dx.doi.org/10.1099/vir.0.007377-0.

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The potential risk of cross-species transmission of porcine endogenous retroviruses (PERV) to humans has slowed the development of xenotransplantation, using pigs as organ donors. Here, we show that PERVs are insensitive to restriction by divergent TRIM5α molecules despite the fact that they strongly restrict a variety of divergent lentiviruses. We also show that the human PERV A/C recombinant clone 14/220 reverse transcribes with increased efficiency in human cells, leading to significantly higher infectivity. We conclude that xenotransplantation studies should consider the danger of highly infectious TRIM5α-insensitive human-tropic PERV recombinants.

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Hsu, Eric, Timothy Osslund, Rebecca Nybo, Bao-Lu Chen, William C. Kenney, C. Fred Morris, Tsutomu Arakawa, and Linda O. Narhi. "Enhanced stability of recombinant keratinocyte growth factor by mutagenesis." Protein Engineering, Design and Selection 19, no. 4 (February 14, 2006): 147–53. http://dx.doi.org/10.1093/protein/gzj013.

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FRATAMICO, PINA M., MING Y. DENG, TERENCE P. STROBAUGH, and SAMUEL A. PALUMBO. "Construction and Characterization of Escherichia coli O157:H7 Strains Expressing Firefly Luciferase and Green Fluorescent Protein and Their Use in Survival Studies‡." Journal of Food Protection 60, no. 10 (October 1, 1997): 1167–73. http://dx.doi.org/10.4315/0362-028x-60.10.1167.

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The firefly (Photinus pyralis) luciferase (luc) gene on plasmid vector pBESTluc and the Aequorea victoria green fluorescent protein (gfp) gene on plasmid vector pGFP were introduced into strains of Escherichia coli O157:H7. The recombinant E. coli strains were indistinguishable from their parent strains in biochemical and immunological assays and in a multiplex PCR reaction. There was no significant difference in the growth kinetics of the luc-bearing recombinants and the parent strains. At 37°C all of the recombinant strains maintained the vectors and expressed luciferase and the green fluorescent protein when grown both with and without antibiotic selection. Individual colonies of luc-bearing E. coli strains were readily luminescent in the dark after being sprayed with a solution of 1 mM beetle luciferin. The recombinants containing pGFP emitted bright green fluorescence when excited with UV light and the addition of any other proteins, substrates, or cofactors was not required. The green fluorescent protein-expressing E. coli O157:H7 strains were used in studies examining the survival of the organism in apple cider and in orange juice. In apple cider the organism declined to undetectable levels in 24 days at refrigeration temperature while in orange juice the strains survived with only small decreases in number during the 24-day sampling period. These recombinant E. coli O157:H7 strains, containing readily identifiable and stable markers, could be useful as positive controls in microbial assays as well as in studies monitoring bacterial survival and the behavior of E. coli O157:H7 in foods and in a food processing environment.

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Chen, Wei, Caiqian Zhang, Yeqing Wu, and Xiuping Su. "Soluble expression and purification of high-bioactivity recombinant human bone morphogenetic protein-2 by codon optimisation in Escherichia coli." Protein Engineering, Design and Selection 32, no. 3 (March 2019): 153–57. http://dx.doi.org/10.1093/protein/gzz028.

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Abstract We developed a simple method of preparing recombinant human bone morphogenetic protein-2 (rhBMP-2) with high biological activity. This rhBMP-2 was overproduced in Escherichia coli as a fusion protein with thioredoxin 6xHis-tag at its amino terminus. The cDNA fragment of human bone morphogenetic protein-2 (hBMP-2) fused to the secretion signal of alkaline phosphatase (PhoA) was expressed under T7 promoter in E. coli. After DNA sequence confirmation, the recombinant vector pETpho-bmp2 was transformed into E. coli BL21 (DE3). rhBMP-2 was produced by the recombinant strain pETpho-bmp2/BL21 (DE3) in a soluble form with an yield of 6.2 mg/L culture. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) results showed that the molecular weight of the product was approximately 28 kD. Moreover, rhBMP-2 was secreted as a dimer with a natural structure. rhBMP-2, purified by Ni Nitrilotriacetic acid Agarose (Ni-NTA) affinity chromatography, was used to examine osteosarcoma MG-63 cells and assay the alkaline phosphatase (ALP) activity. Results showed that rhBMP-2 induced MG-63 cell differentiation. When the final concentration was 500 ng/mL, the effect was more remarkable and ALP activity reached 525% compared with that of the control group.

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Recuenco, Mariam, Md Motiur Rahman, Yoichi Sakamoto, Fusako Takeuchi, Hiroshi Hori, Sam-Yong Park, and Motonari Tsubaki. "1P101 1G1310 FUNCTIONAL CHARACTERIZATION OF THE RECOMBINANT HUMAN TUMOR SUPPRESSOR 101F6 PROTEIN, A CYTOCHROME B561 HOMOLOGUE(Heme proteins,Oral Presentations,The 48th Annual Meeting of the Biophysical Society of Japan)." Seibutsu Butsuri 50, supplement2 (2010): S37. http://dx.doi.org/10.2142/biophys.50.s37_1.

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Banfield, Bruce W., Jessica D. Kaufman, Jessica A. Randall, and Gary E. Pickard. "Development of Pseudorabies Virus Strains Expressing Red Fluorescent Proteins: New Tools for Multisynaptic Labeling Applications." Journal of Virology 77, no. 18 (September 15, 2003): 10106–12. http://dx.doi.org/10.1128/jvi.77.18.10106-10112.2003.

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ABSTRACT The transsynaptic retrograde transport of the pseudorabies virus Bartha (PRV-Bartha) strain has become an important neuroanatomical tract-tracing technique. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV-Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful method for defining connections of single neurons to multiple neural circuits in the brain. However, the present use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods limits the potential of these recombinant virus strains as useful reagents. We previously constructed and characterized PRV152, a PRV-Bartha derivative that expresses the enhanced green fluorescent protein. The development of a strain isogenic to PRV152 and differing only in the fluorescent reporter would have great utility for dual transsynaptic tracing. In this report, we describe the construction, characterization, and application of strain PRV614, a PRV-Bartha derivative expressing a novel monomeric red fluorescent protein, mRFP1. In contrast to viruses expressing DsRed and DsRed2, PRV614 displayed robust fluorescence both in cell culture and in vivo following transsynaptic transport through autonomic circuits afferent to the eye. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; the use of strain PRV614 in combination with strain PRV152 will eliminate many of the pitfalls associated with the presently used pairs of PRV recombinants.

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Journal articles on the topic 'ProteÃna recombinante'

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