Protéines difficiles à exprimer: un goulot d'étranglement pour la plupart des biologistes. J'ai choisi d'utiliser comme modèle d’étude Acanthamoeba polyphaga Mimivirus. Ce virus géant à ADN possède des protéines subissant des modifications post-traductionnelles, des structures multi-protéiques ou encore des voies enzymatiques jamais identifiées auparavant dans un virus, ce qui en font un modèle idéal pour l’étude de protéines récalcitrantes. Le but ultime de cette thèse, était de produire les protéines de capsides de Mimivirus. Le rôle de la protéine de capside dans l’assemblage de la particule virale, son infectivité et ses caractéristiques moléculaires sont d’une grande importance. Pour aller du gène à la protéine, J’ai participé à la compréhension de ce qui gouverne la terminaison de la transcription de Mimivirus et également participé à l'analyse globale du transcriptome au cours du cycle d'infection des amibes par Mimivirus. Nous avons montré que les transcrits de Mimivirus sont systématiquement polyadénylés dans des régions formant une structure secondaire en tige-boucle, même s’il n’existe pas de signal de polyadénylation canonique en amont. Nous en avons conclu que la polyadénylation de Mimivirus suit exclusivement une règle «épingle à cheveux». De plus, l’étude du transcriptome a révélé 3 phases temporelles distinctes dans le cycle infectieux: précoce, intermédiaire et tardive. Les transcrits de capsides sont tous exprimés durant la phase tardive mais leur profil d’expression ne sont pas superposables dans le temps. Les données de transcriptomique ont révélées la présence de plusieurs glycosyltransférases chez Mimivirus, dans la phase tardive du cycle, concomitant avec la production de la protéine de capside. Les informations recueillies sur l'expression des gènes à différents temps post-infection ont contribué à la conception de protocoles pour la production des protéines de capsides (la protéine majeure de capside (MCP) et ses paralogues) dans de systèmes eucaryote. / Difficult to express proteins: a bottleneck for most biologists. I have chosen to use Acanthamoeba polyphaga Mimivirus as my study model. This giant dsDNA virus possesses post-translationally modified proteins, multi-protein structures and enzyme pathways never before seen in a virus, which makes it ideal for refractory studies. The ultimate goal of my thesis was to produce the capsid proteins of Mimivirus. The role of the capsid protein in the assembly of the viral particle, its infectivity, and molecular features are of great importance. To go from gene to protein, I participated in the comprehension of what governs the post-transcriptional termination in Mimivirus and equally participated in the global analysis of the transcriptome during the infectious cycle of Acanthamoeba by Mimivirus. We have shown that the Mimivirus transcripts are systematically polyadenylated in the regions forming a stem-loop secondary structure; even when a canonical poyadenylation signal is absent We concluded that Mimivirus polyadenylation obeys a strict “Hairpin rule”. Moreover, the transcriptomic study revealed three distinct temporal phases: early, intermediate and late. The capsid transcripts are all expressed during the late phase but their expression profiles are not superimposable. The transcriptomic data also revealed the presence of several Mimivirus glycosyltransferases in the late temporal phase, concomitant with the capsid proteins. The expression data gathered throughout my thesis has contributed to the rational design of a protein production experiment to produce the major capsid protein and its three paralogs in eukaryotic systems.

O câncer cervical é um dos tipos de câncer mais comuns entre as mulheres, e a infecção persistente pelos HPV-16 e HPV-18 é responsável por 70% dos casos. As vacinas profiláticas disponíveis possuem alta eficácia na prevenção da infecção pelos tipos mais prevalentes de HPV. No entanto, este tipo de abordagem não beneficia mulheres que já apresentam lesões precursoras ou tumores cervicais avançados, e a busca por abordagens terapêuticas para esse tipo de câncer é considerada uma necessidade. A qualidade do antígeno representa um aspecto fundamental para o sucesso de vacinas terapêuticas baseadas em proteínas recombinantes. Neste sentido, os sistemas de expressão em células eucarióticas, como leveduras e células de mamíferos são considerados adequados para a produção de proteínas com aplicação biotecnológica. O objetivo principal deste trabalho contemplou a expressão das proteínas de fusão gDE7E6 do HPV-16 e do HPV-18 e a oncoproteína E7 do HPV-16 em células da levedura Pichia pastoris e expressão da gDE7E6 do HPV-16 e do HPV-18 em células de mamífero HEK293T e CHODG-44 para obtenção de antígenos purificados com futura aplicação em vacinas terapêuticas contra tumores associados ao HPV-16 e HPV-18. Os genes que codificam as proteínas gDE7E6 dos HPV-16 e HPV-18 e da E7 do HPV-16 foram clonados no vetor pPIC9K, os quais foram linearizados por digestão enzimática e utilizados na transformação da P. pastoris. A expressão das proteínas foi analisada nos tempos de 24, 48, 72 e 96 horas, no entanto, não foi observada a produção das proteínas no sobrenadante e nem no lisado celular. Diante desta constatação, iniciamos a expressão das proteínas gDE7E6 do HPV-16 e gDE7E6 do HPV-18 em células de mamíferos HEK293T e CHODG-44. As sequências genéticas das proteínas gDE7E6 do HPV-16 e do HPV-18 foram clonadas no vetor de expressão pNU1 e analisadas por digestão enzimática. Análises de SDS-PAGE e western blot demonstraram a expressão das proteínas gDE7E6 do HPV-16 e do HPV-18 em até 96 horas em células HEK293T. Em paralelo, realizamos a transfecção estável dos plasmídeos contendo as sequencias da gDE7E6 do HPV-16 e gDE7E6 do HPV-18 em células CHO-DG44. Com o intuito de aumentar a expressão das proteínas de interesse na população mista de CHODG-44, realizamos amplificação genômica com metotrexato (MTX), sendo possível observar aumento da expressão das proteínas, conforme aumento gradativo nas concentrações de MTX. Posteriormente, foram feitas tentativas para isolar um clone produtor das proteínas gDE7E6 HPV-16 e HPV-18, através de clonagem por diluição limitante e sistema automatizado, sendo possível isolar um clone para cada construção através de matriz semisólida, confirmado por western blot e citometria de fluxo. Apesar de demonstrar a expressão das proteínas de interesse em sistema de expressão baseado em células de mamífero, o rendimento obtido após a purificação por afinidade ao níquel foi extremamente baixo, o que dificulta a obtenção dos antígenos para fins vacinais. / Cervical cancer is one of the most common cancers among women, and persistent infection with HPV-16 and HPV-18 accounts for 70% of the cases. Available prophylactic vaccines are highly effective in preventing infection by the most prevalent types of HPV. However, this type of approach does not benefit women who already have precursor lesions or advanced cervical tumors, and the search for therapeutic approaches to this type of cancer is considered a necessity. Antigen quality represents a key aspect for the success of therapeutic vaccines based on recombinant proteins. In this sense, expression systems based in eukaryotic cells such as yeast and mammalian cells are considered suitable for the production of proteins with biotechnological applications. The main objective of this work was to express the gDE7E6 fusion proteins HPV-16 and HPV-18 and the E7 oncoprotein HPV-16 in Pichia pastoris and expression of gDE7E6 HPV-16 and HPV-18 in mammalian cells HEK293T and CHODG-44 to obtain purified antigens with future applications in therapeutic vaccines against HPV-16 and HPV-18 associated tumors. The genes encoding the gDE7E6 proteins HPV-16 and HPV-18 and E7 HPV-16 were cloned into the pPIC9K vector, which were linearized by enzymatic digestion and used in the transformation of P. pastoris. Expression of the proteins was analyzed at 24, 48, 72 and 96 hours, however, the production of the proteins in the supernatant and in the cell lysate was not observed. In light of this finding, we initiated the expression of gDE7E6 proteins HPV-16 and HPV-18 in mammalian cells HEK293T and CHODG-44. The genetic sequences of gDE7E6 proteins HPV-16 and HPV-18 were cloned into the pNU1 expression vector and analyzed by enzymatic digestion. SDSPAGE and western blot analyzes demonstrated expression of gDE7E6 proteins HPV-16 and HPV-18 within 96 hours in HEK293T cells. In parallel, we performed stable transfection of plasmids containing gDE7E6 HPV-16 and HPV-18 sequences into CHODG44 cells. In order to increase the expression of the proteins in the mixed population of CHODG-44, we performed genomic amplification with methotrexate (MTX), and it was possible to observe an increase in protein expression, as a gradual increase in MTX concentrations. Therefore, attempts were made to isolate a clone producing gDE7E6 proteins HPV-16 and HPV-18 by limiting dilution and automated system, being possible to isolate one clone for each construct through a semisolid matrix, confirmed by western blot and flow cytometry. Despite observing protein expression in mammalian cell-based expression system, the yield obtained after nickel affinity purification was extremely low, which makes it difficult to obtain the antigens for vaccine purposes.

No description available.

Transgenic plant systems have been proposed as bioreactors in the production of pharmaceutical and industrial proteins. The economic benefits of inexpensive plant production systems could be erased if the downstream processing ends up being expensive. To avoid monoclonal antibody (mAb) modification or fouling of chromatography resins, removal of phenolics from plant extracts is desirable. Removal of major phenolics in Lemna extracts was evaluated by adsorption to PVPP, XAD-4, IRA-402 and Q-Sepharose resins. Analysis of phenolics adsorption to XAD-4, IRA-402 and Q-Sepharose showed superior dynamic binding capacities at pH 4.5 than at 7.5. The economic analysis using SuperPro Designer 7.0 indicated that addition of a phenolics adsorption step would increase mAb production cost only 20% by using IRA-402 compared to 35% for XAD-4 resin. The overall mAb processing cost can be reduced by implementing a phenolics removal step. To understand phenolics-resin interactions, adsorption isotherms of phenolic compounds (chlorogenic acid, ferulic acid, rutin, syringic acid and vitexin-2-O-rhamnoside) from different phenolic classes on three resins (IRA-402, PVPP, XAD-4) at pH 4.5 and 7.5 were determined. Differences in adsorption with the type of phenolics were observed, and PVPP was not efficient for phenolics removal. Screening of sugarcane lines for bovine lysozyme (BvLz) accumulation indicated that expression levels are still inadequate for commercial development. To maximize BvLz extraction, pH and ionic strength were evaluated; five conditions resulted in equivalent BvLz/TSP ratio. Membrane filtration process using BvLz extracts attained partial removal of native proteins by the 100 kDa membrane step, but also BvLz loss (21-29%). Regardless of the extraction condition, at least 47% of the starting BvLz was lost during the membrane processing. None of the evaluated extraction conditions caused a substantial recovery of BvLz in the concentrate. Alternative purification options for the IEX+HIC process, which achieved 95% BvLz purity, were tested. Direct loading of sugarcane extract concentrate on HIC and XAD-4 pretreatment of juice did not recovered BvLz as effectively as the IEX chromatography. Pure BvLz was obtained by the XAD+HIC process, but higher purification fold and HIC yield were achieved by the IEX+HIC process, due to the complete separation of BvLz and 18-kDa protein.

Plants are potential sources of valuable recombinant and native proteins that can be purified for pharmaceutical, nutraceutical, and food applications. Transgenic rice and corn germ were evaluated for the production of novel protein products. This dissertation addresses: 1) the extraction and purification of the recombinant protein, human lysozyme (HuLZ), from transgenic rice and 2) the processing of dry-milled corn germ for the production of high protein germ and corn protein concentrate (CPC). The factors affecting the extraction and purification of HuLZ from rice were evaluated. Ionic strength and pH was used to optimize HuLZ extraction and cation exchange purification. The selected conditions, pH 4.5 with 50 mM NaCl, were a compromise between HuLZ extractability and binding capacity, resulting in 90% purity. Process simulation was used to assess the HuLZ purification efficiency and showed that the processing costs were comparable to native lysozyme purification from egg-white, the current predominant lysozyme source. Higher purity HuLZ (95%) could be achieved using pH 4.5 extraction followed by pH 6 adsorption, but the binding capacity was unexpectedly reduced by 80%. The rice impurity, phytic acid, was identified as the potential cause of the unacceptably low capacity. Enzymatic (phytase) treatment prior to adsorption improved purification, implicating phytic acid as the primary culprit. Two processing methods were proposed to reduce this interference: 1) pH 10 extraction followed by pH 4.5 precipitation and pH 6 adsorption and 2) pH 4.5 extraction and pH 6 adsorption in the presence of TRIS counter-ions. Both methods improved the binding capacity from 8.6 mg/mL to >25 mg/mL and maintained HuLZ purity. Processing of dry-milled corn germ to increase protein and oil content was evaluated using germ wet milling. In this novel method, dry-milled germ is soaked and wet processed to produce higher value protein products. Lab-scale and pilot-scale experiments identified soaking conditions that reduced germ starch content, enhanced protein and oil content, and maintained germ PDI (protein dispersibility index). Soaking at neutral pH and 25 degrees C maintained germ PDI and improved CPC yield from defatted germ flour. CPC with greater than 75% protein purity was produced using protein precipitation or membrane filtration.

No description available.

<p>The aim of this work was to investigate and determine central parameters that can be used to control and increase the solubility, quality and productivity of recombinant proteins. These central parameters should be applicable under the constraints of high-throughput protein production in <em>Escherichia coli.</em></p><p>The present investigation shows that alternative methods exist to improve solubility, quality and productivity of the recombinant protein. The hypothesis is that by reducing the synthesis rate of the recombinant protein, a higher quality protein should be produced. The feed rate of glucose can be used to decrease the synthesis rate of the recombinant protein.</p><p>The influence of feed rate on solubility and proteolysis was investigated using the <em>lac</em>UV5-promoter and two model proteins, Zb-MalE and Zb-MalE31. Zb-MalE31 is a mutated form of Zb-MalE that contains two different amino acids. These altered amino acids greatly affect the solubility of the protein. The soluble fraction is generally twice as high using Zb-MalE compared to Zb-MalE31. Using a low feed rate compared to high benefits the formation of the full-length soluble protein. Furthermore, by using a low feed rate, the proteolysis can be decreased. One other factor that influences the solubility is the amount of inducer used. An increase from 100 µM to 300 µM IPTG only results in more inclusion bodies being formed, the fraction of soluble protein is the same.</p><p>The quality aspect of protein production was investigated for a secreted version of Zb-MalE using two different feed rates of glucose and the maltose induced promoter P<em>malK</em>. It was shown that when the protein was secreted to the periplasm, the stringent response as well as the accumulation of acetic acid (even for high feed rates) was reduced. The stringent response and accumulation of acetic acid are factors that are known to affect the quality and quantity of recombinant proteins. Transporting the protein to the periplasm results in this case on a lower burden on the cell, which leads to less degradation products being formed when the protein is secreted to the periplasm.</p><p>Seeing the feed rate as a critical parameter, the high-throughput production would benefit from a variation in the feed rate. However, since the fed-batch technique is technically complicated for small volumes another approach is needed. <em>E.coli</em> strains that have been mutated to create an internal growth limitation that simulate fed-batch were cultivated in batch and were compared to the parent strain. It was shown that the growth rate and acetic acid formation was comparable to the parent strain in fed-batch. Furthermore it was shown that a higher cell mass was reached using one of the mutants when the cells were cultivated for as long time as possible. The higher cell mass can be used to reach a higher total productivity.</p>

Drug resistant strains of Clostridium difficile are a major health concern with over 3 million cases costing over 1 billion $ per year in the United-States. The diseases associated with these bacteria (CDAD) are toxin-mediated which offers a mean of treating and lessening the severity of CDAD symptoms. Toxin inactivation via antibodies therapy can drastically reduce CDAD morbidity and this project was aiming at investigating the large-scale production and recovery of a novel llama single domain antibody (pSJF2H-ToxA5.1) in recombinant Escherichia coli TG1 targeting C. difficile enterotoxin A (TcdA). In order to achieve these objectives, the project was divided into four segments: 1) ToxA5.1 being an intracellular recombinant protein, obtaining a high biomass production was the first step towards large-scale production. To achieve HCDC, effects of initial glucose concentration and pH-stat feeding strategy were studied; 2) Upon achieving HCDC, effects of parameters such as temperature, induction timing and media supplementation with complex nitrogen sources were investigated; 3) Once large-scale production of ToxA5.1 was obtained, the recombinant protein needed to be recovered and a selective cell lysis scheme where synergistic lysis effects of Triton X-100 and temperature were studied. And finally 4) Single-step purification using nickel nanoparticles (NNP) synthesized via a modified polyol method was studied. Combining the HCDC strategy with a temperature shift and yeast extract addition at the time of induction, ToxA5.1 concentration of 127 mg/L was obtained. Synergistic and selective cell lysis using Triton X-100 and temperature was achieved where 95% of the available ToxA5.1 was recovered and still functional while ToxA5.1 fraction in the resulting lysate increased to 27% in the cell lysate. Single-step purification was achieved using the synthesized NNP which proved to be highly selective and could be used up to five times. Diameter of the NNP synthesized was controlled by using various concentration of ranging from 131 ± 80 nm to 47 ± 20 nm. Using experimental data from binding isotherm, the ToxA5.1-NNP system was modeled.

Aeromonas hydrophila is an opportunistic pathogen that causes a wide range of symptoms and diseases in fish. Development of a commercial vaccine has been problematic due to the heterogenicity between isolates of A. hydrophila. A new approach using immunoproteomics was used in this study to try to develop a vaccine that would protect against a wide range of A. hydrophila strains. The virulence of 14 isolates of A. hydrophila from different geographical regions was determined in common carp (Cyprinus carpio) indicating that 6 isolates were virulent, while 8 isolates were avirulent. Expression of cellular and extracellular products (ECP) of six of these isolates (4 virulent and 2 avirulent isolates) were examined following culture of the bacterium in vitro, in tryptic soy broth, and in vivo, in dialysis tubing placed within the peritoneal cavity of carp. Two types of molecular weight cut off tubes (25 and 100 kDa) were used for the implants. Whole cell (WC), outer membrane protein (OMP) and ECPs of the bacteria grown in vitro and in vivo were analysed by 1 dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE). Additionally, 2D SDS-PAGE was used to analyse WC preparations of A. hydrophila grown in vitro and in vivo. The production of unique proteins and up and down-regulation of protein expression were observed in all the preparations of bacteria grown in vitro and in vivo. Unique bands were seen in the 1D SDS-PAGE at 58 and 55 kDa for WC and OMP preparations, respectively, for all the isolates cultured in vivo. Bands of increased intensity were observed at 70, 55, 50 and 25 kDa with WC preparations for the virulent isolates cultured in vivo. Analysis of WC preparations by 2D SDS-PAGE indicated differences in the expression of spots between bacteria cultured in vitro and in vivo. A number of unique spots, mostly between 30 and 80 kDa with pI values ranging from 5.0-6.0 were observed in the bacteria grown in vivo. The protein profiles of different preparations (WC, OMP, ECP) of bacteria cultured in vitro and in vivo were screened by 1D Western blot using antibodies from carp artificially infected with different isolates of A. hydrophila to identify potential vaccine candidates. The WC preparations of A. hydrophila (T4 isolate) grown in vitro were also analysed by 2D Western blot. A 50 kDa protein of A. hydrophila was found to be the most immunogenic molecule in both WC and OMP of bacteria grown both in vitro and in vivo. The protection efficacy of this protein was determined in goldfish by vaccinating fish with electro-eluted 50 kDa protein then challenging the fish with A. hydrophila. Fish were also passively immunised with fish sera raised to the 50 kDa protein and then challenged. The relative percentage survival (RPS) was 67 % in the vaccination trial, while the results were inconclusive for the passive immunisation trial. The 50 kDa protein was confirmed to be the S-layer protein of A. hydrophila following identification using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Recombinant S-layer protein was then produced and the cross-protection efficacy of this protein against six virulent isolates of A. hydrophila was confirmed in a large scale vaccination trial using carp. The RPS value for the 6 isolates of A. hydrophila ranged from between 56 and 87 %. The results of this project suggest that the immunogenic S-layer protein of A. hydrophila could be used as a common antigen to protect fish against infection by different isolates of this pathogenic bacterium.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-20Bitstream added on 2014-06-13T18:44:31Z : No. of bitstreams: 1 gibertoni_am_dr_jabo.pdf: 1047291 bytes, checksum: 9be7492afd065b969b043d3a6d16e4be (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Foram realizadas a clonagem e expressão do gene da nucleoproteína (N) de uma estirpe vacinal de referência M41 do vírus da bronquite infecciosa (VBI), como proteína recombinante de fusão, contendo uma cauda de poli-histidina na extremidade carboxi-terminal, em 2 sistemas hospedeiros; na levedura metilotrófica, Pichia pastoris e na bactéria Escherichia coli. A proteína N derivada de um isolado variante do VBI de surtos a campo no Brasil, também foi expressa em E. coli. As características bioquímicas e imunoquímicas de tais proteínas recombinantes, foram determinadas, tendo sido evidenciado maior eficiência de produção no sistema hospedeiro constituído por E. coli, comparativamente ao sistema composto por P. pastoris. Uma vez obtidas, caracterizadas e purificadas, através da técnica de cromatografia de afinidade em resina de níquel-sepharose, as preparações de proteína N recombinante expressas em E. coli e derivadas ou da estirpe de referência M41 ou do novo isolado de campo no Brasil, foram utilizadas de forma bem sucedida, como antígenos alvo de ensaios indiretos de ELISA, que foram aplicados na detecção e mensuração de anticorpos dos isótipos IgG e IgM em aves infectadas com estirpes homóloga ou variantes do VBI. Foi, também, investigada a atividade imunogênica da proteína N recombinante em aves, que depois de imunizadas e re-imunizadas com essas proteínas recombinantes, produziram no soro sanguíneo e na secreção lacrimal quantidades elevadas de anticorpos anti-VBI específicos, mas não desenvolveram proteção efetiva contra o desafio com a estirpe homóloga desse vírus. Concluindo, a proteína N recombinante do VBI expressa pela E. coli possui elevada imunogenicidade, no sentido de induzir altos níveis de anticorpos específicos, e reatividade cruzada com proteínas N de outras variantes desse vírus, tendo um grande potencial de ser aplicada em... / Two host systems, represented by Escherichia coli and Pichia pastoris were used for cloning and protein expression of the nucleoprotein (N) gene of M41 strain of infectious bronchitis virus (IBV) as a fusion recombinant protein containing a poli-histidine tag. The N protein from a new variant Brazilian field isolate was also cloned and expressed by E. coli system. The biochemical and immunochemical properties of these recombinant N proteins were determined and higher efficiency on protein production was achieved by using the E. coli expression system. Both recombinant N proteins expressed by E. coli were purified in nickel-sepharose resin and used as antigen in indirect ELISA methods for the detection of IgG and IgM antibodies in birds infected with homologous and variant IBVs. The immunogenicity of N recombinant protein was also evaluated by immunizing and re-immunizing birds and high antibody levels were generated in lachrymal secretion and serum, but no effective protection against challenge with homologous virulent stain of IBV was induced. Concluding, the recombinant N IBV protein expressed by E. coli is highly immunogenic for inducing specific and crossreactive antibodies, and can be applied in the immuno-diagnosis of IB