Rozprawy doktorskie: „Escherichia coli Inclusions”

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RODRIGUES, DANIELLA. "Utilização de altas pressões hidrostáticas para o estudo e renaturação de proteínas com estrutura quaternária". reponame:Repositório Institucional do IPEN, 2012. http://repositorio.ipen.br:8080/xmlui/handle/123456789/10161.

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Dissertação (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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Wangsa-Wirawan, Norbertus Djajasantosa. "Physicochemical properties of protein inclusion bodies". Title page, contents and introduction only, 1999. http://web4.library.adelaide.edu.au/theses/09PH/09phw2465.pdf.

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Bibliography: leaves 182-198. Improvements in the current production system of inclusion bodies and the downstream processing sequence are essential to maintain a competitive advantage in the market place. Optimisation of fermentation is considered to improve production yield; then flotation as a possible inclusion body recovery method.

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BALDUINO, KELI N. "Renaturacao em altas pressoes hidrostaticas de proteinas recombinantes agregadas em corpos de inclusao produzidos em Eschirichia coli". reponame:Repositório Institucional do IPEN, 2009. http://repositorio.ipen.br:8080/xmlui/handle/123456789/9457.

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IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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Wong, Heng Ho. "Modelling studies of the interaction between homogenisation, centrifugation and inclusion body dissolution /". Title page, contents and summary only, 1997. http://web4.library.adelaide.edu.au/theses/09PH/09phw8718.pdf.

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Saulou, Claire. "Evaluation des propriétés anti-adhésives et biocides de films nanocomposites avec inclusions d’argent, déposés sur acier inoxydable par procédé plasma". Toulouse, INSA, 2009. http://eprint.insa-toulouse.fr/archive/00000315/.

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Dans le secteur biomédical et l’industrie agro-alimentaire, l’adhésion de microorganismes contaminants aux surfaces engendre de multiples impacts négatifs, à la fois en termes de santé publique, d’hygiène et de sécurité alimentaire. Dans ce contexte, l’objectif de l’étude est de mettre au point un traitement de surface de l’acier inoxydable 316L, afin de prévenir la colonisation microbienne. La modification des surfaces d’acier par traitement chimique ou physique n’a eu aucune incidence sur le détachement de Saccharomyces cerevisiae, évalué in vitro à l’aide d’une chambre à écoulement cisaillé. Les interactions entre la surface microbienne et les éléments métalliques du film passif semblent jouer un rôle prépondérant dans cette forte adhésion. Une stratégie originale, basée sur un procédé plasma couplant la polymérisation d’hexaméthyldisiloxane au bombardement d’une cible d’argent dans une décharge asymétrique radiofréquence, a ensuite été mise en œuvre et optimisée. Les surfaces d’acier ont ainsi été recouvertes de films minces (~ 175 nm) nanocomposites, constitués d’une matrice organosiliciée, présentant des propriétés anti-adhésives vis-à-vis de S. Cerevisiae, dans laquelle ont été incluses des nanoparticules d’argent, dotées d’une forte réactivité antimicrobienne. Le couplage de techniques d’analyse complémentaires, opérant à différentes échelles, a permis de corréler les caractéristiques des films nanocomposites à leur efficacité anti-adhésive et antifongique. Une inhibition totale de l’adhésion des levures a ainsi été obtenue, en augmentant le caractère polaire de la matrice, par ajout d’oxygène dans le plasma. En parallèle, un abattement de la viabilité de 1,9 log a été atteint sur les levures sessiles. La suite de l’étude a été dédiée à la compréhension des mécanismes d’action de l’argent, impliqués dans l’activité antifongique des films nanocomposites. Une inactivation de certaines protéines pariétales et intracellulaires, corrélée à des altérations de l’ultra-structure cellulaire, a ainsi été mise en évidence. La confirmation de l’activité biocide des films nanocomposites, sur deux modèles procaryotes (Staphylococcus aureus et Escherichia coli), a révélé par ailleurs la nécessité d’un contact étroit entre microorganismes et revêtement. Enfin, la stabilité des propriétés des films nanocomposites a été évaluée. Une utilisation répétée des dépôts a mis en évidence une réduction de l’activité antifongique, corrélée à une augmentation de l’efficacité anti-adhésive, liée au relargage d’argent lors de la première utilisation
In the biomedical domain and the food industry, microbial adhesion to surfaces generates multiple negative consequences, in terms of human health, hygiene and safety of processed food. In this context, our approach is based on developing a 316L stainless steel surface treatment, to prevent microbial colonization. The surface modifications, mediated by chemical or physical treatment, did not promote Saccharomyces cerevisiae detachment, evaluated in vitro using a shear stress flow chamber. The interactions between the microbial surface and metallic elements of the passive film were hypothesized to play a predominant role in this strong adhesion. An original and dual strategy, based on a plasma process associating hexamethyldisiloxane polymerization and silver target bombardment in an asymmetrical radiofrequency discharge, was carried out and optimized. Stainless steel surfaces were thus coated with nanocomposite thin films (~ 175 nm), composed of an organosilicon matrix, exhibiting anti-adhesive properties towards S. Cerevisiae, in which were embedded silver nanoparticles, displaying a high antimicrobial reactivity. A large set of complementary analytical techniques, operating at different scales, was used to correlate nanocomposite film characteristics with their anti-adhesive and antimicrobial efficiency. A total inhibition of yeast cell adhesion was achieved, by increasing the matrix polar character, through oxygen addition during the plasma process. In parallel, a 1. 9 log reduction in viable counts was achieved for sessile yeast cells. Further experiments were dedicated to the thorough understanding of cellular changes induced by silver release. A deterioration of the secondary structure of proteins (cell wall, intracellular), combined with ultra-structure alterations, was observed. In addition, the biocide activity of the nanocomposite film was confirmed against two prokaryotic models (Staphylococcus aureus and Escherichia coli). The necessity of a direct contact between microorganisms and coating was demonstrated for a maximal antimicrobial efficiency. Lastly, the durability of the coating properties was assessed through a repeated use of the nanocomposite films. A decrease in the antifungal activity, coupled to an anti-adhesive property enhancement, was noticed and explained by the silver release during the first use

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Hart, Roger A. Bailey James E. Bailey James E. "Characterization of Vitreoscilla hemoglobin inclusion bodies produced in Escherichia coli /". Diss., Pasadena, Calif. : California Institute of Technology, 1991. http://resolver.caltech.edu/CaltechETD:etd-06272007-152616.

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Olbrich, Richard. "The characterisation and recovery of protein inclusion bodies from recombinant Escherichia-coli". Thesis, University College London (University of London), 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324583.

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Garcia, i. Fruitós Elena. "Regulation of recombinant proteína solubility and conformational quality in Escherichia coli". Doctoral thesis, Universitat Autònoma de Barcelona, 2008. http://hdl.handle.net/10803/3923.

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All the processes that take place in a cell require one or more proteins, meaning that they are essential components of life. Proteins are macromolecules consisting of amino acid units, all of them being constructed with combinations of only 20 amino acids. The primary structure of a protein molecule is determined by the sequence of amino acids connected by peptide bonds forming a polypeptide chain. Once the amino acid chain is synthesized, the protein folds by a physical process that might be eventually assisted by other proteins, reaching its characteristic three‐dimensional structure that is the final, functional conformation. However, although it is known that all the proteins must properly fold into their correct native conformation to be functional, their final conformation cannot be predicted from their primary amino acid sequence, being protein folding mechanisms one of the most challenging problems in biology today. Many proteins of relevant industrial or medical value are produced in low amounts in their natural sources. However, at the end of the seventies, the development of recombinant DNA technologies opened a new promising era for protein production in high amounts for both research and industrial applications. This had a tremendous impact, for example, in many areas of medicine as a tool to produce new drugs for the treatment of diseases and genetic disorders. Genetic engineering permits the introduction of the encoding genes of the protein of interest into recipient cells, where these genes are positioned downstream of regulable promoters in movable genetic elements, mainly plasmids. Under suitable conditions, these transgenic cells acting as protein production bio‐factories would be expected to act as unlimited and inexpensive source of rare, highly valuable proteins not only for proteomics and structural functional genomics1 but also for large‐scale preparative purposes. The quality as well as the quantity of the produced recombinant protein is greatly influenced by the chosen biological cell system. Bacteria have been the most commonly used organisms for protein production, specially the enterobacteria Escherichia coli, not only for the low cost of the used processes, but also for its fast growth. Generally, in Escherichia coli, the rather small host cell proteins can fold properly, adopting a native, biological active conformation. However, when producing heterologous proteins, specially those with eukaryotic or viral origin, important obstacles appear during the protein production process: a) in most cases, the protein is produced in a non functional conformation; b) sometimes the formed product is toxic for the cell; c) the protein often results proteolytically degraded2; d) the product is accumulated as an insoluble, non‐functional protein aggregates, known as inclusion bodies3. Therefore, even though the important advantages of the use of bacteria as a expression system, Escherichia coli also presents some drawbacks, such as its inability to carry most of the post‐transcriptional modifications, often required for eukaryotic protein function, the lack of a secretion mechanism to release the protein to the medium, and the inability to create an oxidative environment to facilitate disulfide bond formation required to achieve the final, functional structure of some proteins. Therefore, this leads to the production of proteins which are not always suitable for immediate use. This means that, to date, many proteins have been excluded from the biotechnological and pharmaceutical market because they cannot be produced in high yields as soluble and active products. To avoid protein folding problems encountered in bacteria under overexpression conditions, mainly secretion and post‐transcriptional modifications, alternative host cells, such as yeast, filamentous fungi, mammalian or insect cells, have been explored. Nevertheless, an enormous number of deficiencies in these systems such as difficulty of genetic manipulation, low productivity and high costs, shows that these organisms are not ideal for this aim and that, even when bacteria show some obstacles in the production process and often this system has to be optimized for specific products, it is, in most of the cases, the best choice.

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Mikkola, Isak. "Does SCP-2 promote the expression of foreign proteins in Escherichia coli?" Thesis, Linköpings universitet, Biologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-129802.

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Expression of foreign proteins in host organisms usually results in the development of insoluble, inactive proteins. Further, these proteins have a tendency to form aggregates termed inclusion bodies. However, the formation of inclusion bodies can be avoided by fusing the gene encoding the foreign protein to a highly soluble protein. In this report Sterol Carrier Protein-2 (SCP-2) is reviewed as a possible solubility tag. The experiment was carried out by fusing SCP-2 to one of two i nsoluble proteins, Green fluorescent protein (GFP) or a form of chloramphenicol acetyl transferase (CAT∆9). The protein fusion was then inserted into the vector pET-15b, transformed in Escherichia coli and the yield of actively expressed protein was measured. The results obtained from this study, as evaluated by PageBlue staining and Western blot, are indicating that SCP-2 does not improve the solubility of GFP or CAT∆9. Nonetheless, the solubility of GFP has earlier been increased by fusing it to the solubility tag maltose-binding protein (MBP). Producing more soluble forms of CAT∆9 have also been tested but without success. Therefore the conclusion drawn from this experiment is that SCP-2 does not work as a solubility tag, however more research must be performed to conclude this with certainty.

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Hedhammar, My. "Strategies for facilitated production of recombinant proteins in escherichia coli". Doctoral thesis, KTH, School of Biotechnology (BIO), 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-471.

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The successful genomic era has resulted in a great demand for efficient production and purification of proteins. The main objective of the work described in this thesis was to develop methods to facilitate recovery of target proteins after recombinant production in Escherichia coli.

A positively charged purification tag, Z_basic, has previously been constructed by protein design of a compact three-helix bundle domain, Z. The charged domain was investigated for general use as a fusion partner. All target proteins investigated could be selectively captured by ion-exchange chromatography under conditions excluding adsorption of the majority of Escherichia coli host proteins. A single cation-exchange chromatography step at physiological pH was sufficient to provide Z_basic fusion proteins of high purity close to homogeneity. Moreover, efficient isolation directly from unclarified Escherichia coli homogenates could also be accomplished using an expanded bed mode. Since the intended use of a recombinant protein sometimes requires removal of the purification tag, a strategy for efficient release of the Z_basic moiety using an immobilised protease was developed. The protease columns were reusable without any measurable decrease in activity. Moreover, subsequent removal of the released tag, Z_basic, was effected by adsorption to a second cation-exchanger.

Using a similar strategy, a purification tag with a negatively charged surface, denoted Z_acid, was constructed and thoroughly characterised. Contrary to Z_basic, the negatively charged Z_acid was highly unstructured in a low conductivity environment. Despite this, all Z_acid fusion proteins investigated could be efficiently purified from whole cell lysates using anion-exchange chromatography

Synthesis of polypeptides occurs readily in Escherichia coli providing large amounts of protein in cells of this type, albeit often one finds the recombinant proteins sequestered in inclusion bodies. Therefore, a high throughput method for screening of protein expression was developed. Levels of both soluble and precipitated protein could simultaneously be assessed in vivo by the use of a flow cytometer.

The positively charged domain, Z_basic, was shown also to be selective under denaturing conditions, providing the possibility to purify proteins solubilised from inclusion bodies. Finally, a flexible process for solid-phase refolding was developed, using Z_basic as a reversible linker to the cation-exchanger resin.

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Hoffmann, Daniel [Verfasser]. "Produktion des Insektenmetalloprotease Inhibitors in Escherichia coli : Neuartige Plattformtechnologie für die inclusion body-basierte Produktaufarbeitung / Daniel Hoffmann". Aachen : Shaker, 2019. http://d-nb.info/1190525623/34.

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Hedhammar, My. "Strategies for facilitated protein recovery after recombinant production in Escherichia coli". Doctoral thesis, KTH, Proteomik, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-471.

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The successful genomic era has resulted in a great demand for efficient production and purification of proteins. The main objective of the work described in this thesis was to develop methods to facilitate recovery of target proteins after recombinant production in Escherichia coli. A positively charged purification tag, Zbasic, has previously been constructed by protein design of a compact three-helix bundle domain, Z. The charged domain was investigated for general use as a fusion partner. All target proteins investigated could be selectively captured by ion-exchange chromatography under conditions excluding adsorption of the majority of Escherichia coli host proteins. A single cation-exchange chromatography step at physiological pH was sufficient to provide Zbasic fusion proteins of high purity close to homogeneity. Moreover, efficient isolation directly from unclarified Escherichia coli homogenates could also be accomplished using an expanded bed mode. Since the intended use of a recombinant protein sometimes requires removal of the purification tag, a strategy for efficient release of the Zbasic moiety using an immobilised protease was developed. The protease columns were reusable without any measurable decrease in activity. Moreover, subsequent removal of the released tag, Zbasic, was effected by adsorption to a second cation-exchanger. Using a similar strategy, a purification tag with a negatively charged surface, denoted Zacid, was constructed and thoroughly characterised. Contrary to Zbasic, the negatively charged Zacid was highly unstructured in a low conductivity environment. Despite this, all Zacid fusion proteins investigated could be efficiently purified from whole cell lysates using anion-exchange chromatography Synthesis of polypeptides occurs readily in Escherichia coli providing large amounts of protein in cells of this type, albeit often one finds the recombinant proteins sequestered in inclusion bodies. Therefore, a high throughput method for screening of protein expression was developed. Levels of both soluble and precipitated protein could simultaneously be assessed in vivo by the use of a flow cytometer. The positively charged domain, Zbasic, was shown also to be selective under denaturing conditions, providing the possibility to purify proteins solubilised from inclusion bodies. Finally, a flexible process for solid-phase refolding was developed, using Zbasic as a reversible linker to the cation-exchanger resin.
QC 20101020

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Boström, Maria. "Design of substrate induced transcription for control of recombinant protein production in Escherichia coli". Doctoral thesis, KTH, Biotechnology, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3834.

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Henderson, Ian. "Solving the inclusion body problem - a case study : high level expression of TEM-1 #beta#-lactamase in Escherichia coli". Thesis, University of Warwick, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.282432.

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Truong, Vy Thuy. "Effect of cinnamic acid-cyclodextrin inclusion complexes on populations of Escherichia coli O157:H7 and Salmonella enterica in fruit juices". Thesis, Virginia Tech, 2007. http://hdl.handle.net/10919/35622.

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Cinnamic acid (CA) is a naturally occurring organic acid that is found in some fruits and a number of spices. CA has antimicrobial activity against certain spoilage microorganisms and pathogenic bacteria. However, the acid is poorly soluble in water. Cyclodextrin molecules have a hydrophobic cavity that allows them to serve as a host for insoluble molecules in aqueous matrices. This study was conducted to determine if the aqueous solubility of cinnamic acid could be improved via complexation with Î±- or Î²-cyclodextrins, and if these complexes could be used to control bacterial pathogens in juices. Based upon phase solubility analysis, Î±-cyclodextrin was chosen as the host molecule for the remainder of this study. In complex with Î±-cyclodextrin, the solubility of cinnamic acid increased from approximately 400 mg/L to 3800 mg/L. Prepared cinnamic acid complexed with Î±-cyclodextrin was aseptically added (400 mg/L and 1000 mg/L) to orange juice inoculated with a Salmonella enterica (7 log CFU/mL) and apple cider inoculated with Escherichia coli O157:H7 (7 log CFU/mL). Cider and orange juice samples were extracted on day 0 and at 24 h intervals for seven days and spread plated onto Tryptic Soy Agar. Cinnamic acid was effective for reducing populations of both bacterial pathogens in juice. Populations of E. coli O157:H7 in the apple cider were significantly reduced after 7 days at 25.6 Â± 0.42Â° C at concentrations of 400 mg/L (5-log CFU/mL reduction) and 1000 mg/L (6-log CFU/mL reduction) cyclodextrin-cinnamic acid. S. enterica counts were also reduced in orange juice at 4Â° C treated with 400 mg/L (2.7-log CFU/mL reduction) and 1000 mg/L (3.2-log CFU/mL reduction) complexed cinnamic acid. The much improved solubility of this compound provides food processors with greater flexibility in using cinnamic acid in their product formulations.
Master of Science

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Gomes, Fernanda Resende. "Expressão do fator estimulador de colônia de granulócito humano recombinante (rhG-CSF) em Escherichia coli". Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-13082010-163827/.

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O Fator estimulador de colônias de granulócitos humano recombinante (rhG-CSF) produzido em Escherichia coli é uma proteína não glicosilada com 175 aminoácidos, de grande importância clínica para o tratamento de neutropenias. O presente trabalho propõe a construção de dois sistemas de expressão em E. coli, um sistema para obtenção do rhG-CSF no citoplasma e outro para secreção da proteína recombinante no meio de cultura utilizando a sequência sinal da L-asparaginase II. Os dois sistemas de expressão foram testados e comparados. A partir desses dados, passou-se para as etapas de obtenção do rhG-CSF com o sistema de expressão sem a sequência sinal. As etapas de renaturação e purificação foram eficientes obtendo-se uma proteína com adequado grau de pureza, integridade estrutural e atividade biológica. Essa proteína também foi utilizada com sucesso para a produção de anticorpos policlonais em camundongos. Com os resultados obtidos, a proteína rhG-CSF mostrou-se viável para estudos posteriores em bioreatores e produção em escala-piloto.
The recombinant human granulocyte colony stimulating factor (rhG-CSF) is a non-glycosylated protein with 175 amino acids. This factor plays an important role in hematopoietic cell proliferation and has been widely used for treating neutropenia. The purpose of this work is to construct two expression systems in E. coli; a system for obtaining rhG-CSF in the cytoplasm and the other for secretion of recombinant protein in the culture medium using the signal sequence of L-asparaginase II. The two expression systems were tested and compared. From these data, the next steps for obtaining the rhG-CSF were done with the expression system without the signal sequence. The refolding and purification steps were efficient, resulting in a protein with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of polyclonal antibodies in mice. With these results, the protein rhG-CSF was feasible for further studies in bioreactors and pilot scale production.

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Le, Thanh Ha. "Optimisation of active recombinant protein production, exploring the impact of small heat-shock proteins of Escherichia coli, IbpA and IbpB, on in vivo reactivation of inclusion bodies". [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=975691554.

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Reitz, Christian [Verfasser], Peter [Akademischer Betreuer] Neubauer, Peter [Gutachter] Neubauer, Vera [Gutachter] Meyer i Ralf [Gutachter] Takors. "Impacts of oscillating cultivation conditions on the quality of recombinant inclusion bodies in Escherichia coli / Christian Reitz ; Gutachter: Peter Neubauer, Vera Meyer, Ralf Takors ; Betreuer: Peter Neubauer". Berlin : Technische Universität Berlin, 2017. http://d-nb.info/1156012856/34.

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Lu, Ping [Verfasser], Peter [Akademischer Betreuer] Neubauer, Peter [Gutachter] Neubauer i Thomas [Gutachter] Schweder. "Response of Escherichia coli processes for the production of heterologous inclusion bodies by oscillating cultivation conditions in a scale-down bioreactor / Ping Lu ; Gutachter: Peter Neubauer, Thomas Schweder ; Betreuer: Peter Neubauer". Berlin : Technische Universität Berlin, 2016. http://d-nb.info/1156016312/34.

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Sundström, Heléne. "Analytical tools for monitoring and control of fermentation processes". Doctoral thesis, KTH, Skolan för bioteknologi (BIO), 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4531.

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The overall objective of this work has been to adopt new developments and techniques in the area of measurement, modelling and control of fermentation processes. Flow cytometry and software sensors are techniques which were considered ready for application and the focus was set on developing tools for research aiming at understanding the relationship between measured variables and process quality parameters. In this study fed-batch cultivations have been performed with two different strains of Escherichia coli (E.coli) K12 W3110 with and without a gene for the recombinant protein promegapoietin. Inclusion body formation was followed during the process with flow cytometric detection by labelling the inclusion bodies with first an antibody against the protein promegapoietin and then a second fluorescent anti-antibody. The approach to label inclusion bodies directly in disintegrated and diluted cell slurry could be adopted as a method to follow protein production during the process, although the labelling procedure with incubation times and washings was somewhat time-consuming (1.5 h). The labelling of inclusion bodies inside the cells to follow protein production was feasible to perform, although an unexplained decrease in the relative fluorescence intensity occurred late in process. However, it is difficult to translate this qualitative measurement into a quantitative one, since a quantitative protein analysis should give data proportional to the volume, while the labelling of the spheric inclusion bodies gives a signal corresponding to the area of the body, and calibration is not possible. The methods were shown to be useful for monitoring inclusion body formation, but it seems difficult to get quantitative information from the analysis. Population heterogeneity analysis was performed, by using flow cytometry, on a cell population, which lost 80-90% viability according to viable count analysis. It was possible to show that the apparent cell death was due to cells incapable of dividing on agar plates after induction. These cells continued to produce the induced recombinant protein. It was shown that almost all cells in the population (≈97%) contained PMP, and furthermore total protein analysis of the medium indicated that only about 1% of the population had lysed. This confirms that the "non-viable" cells according to viable count by cfu analysis produced product. The software sensors XNH3 and µNH3, which utilises base titration data to estimate biomass and specific growth rate was shown to correlate well with the off-line analyses during cultivation of E. coli W3110 using minimal medium. In rich medium the µNH3 sensor was shown to give a signal that may be used as a fingerprint of the process, at least from the time of induction. The software sensor KLaC* was shown to respond to foaming in culture that probably was caused by increased air bubble dispersion. The RO/S coefficient, which describes the oxygen to substrate consumption, was shown to give a distinct response to stress caused by lowered pH and addition of the inducing agent IPTG. The software sensor for biomass was applied to a highly automated 6-unit multi-bioreactor system intended for fast process development. In this way also specific rates of substrate and oxygen consumption became available without manual sampling.
QC 20100819

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Hart, Roger A. "Characterization of Vitreoscilla hemoglobin inclusion bodies produced in Escherichia coli". Thesis, 1991. https://thesis.library.caltech.edu/2745/1/Hart_ra_1991.pdf.

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The process of inclusion body (IB) formation in the gram-negative bacterium Escherichia coli (E. coli) was investigated. The homodimeric hemeprotein Vitreoscilla hemoglobin (VHb) from the gram-negative bacterium Vitreoscilla was taken as the model protein. Expression of VHb under control of its native promoter from a pUC19-derived plasmid in strain JM101 lead to high-level accumulation of VHb in both soluble and insoluble forms. The soluble form was purified by sequential two-phase extraction techniques and used as a basis for analyzing the insoluble form. The amino acid content and N-terminal sequence of purified soluble VHb is consistent with that of VHb purified from Vitreoscilla. Soluble and insoluble VHb exhibit identical migration properties during denaturing two-dimensional electrophoresis. The protein composition of VHb inclusion bodies was analyzed by one-dimensional and two-dimensional electrophoresis techniques. Results indicate the presence of two types of cytoplasmic aggregates of differing morphology in single bacterial cells. These aggregates also differ in their relative content of VHb, pre-[beta]-lactamase, and the cytoplasmic protein elongation factor Tu and are separable by differential centrifugation. Conformational properties of soluble and insoluble VHb were studied by electron paramagnetic resonance spectroscopy. Purified soluble VHb exhibits three high-spin resonances in the vicinity of g 6 from two heme centers. One center is axial (g 6.00). The other is rhombic (g 5.50 and 6.39). Inclusion body isolates containing insoluble VHb exhibit a single resonance (g 5.98) which is also present in control cell debris. Iron quantitation demonstrates that inclusion body VHb uniformly lacks heme. Titration of IB fractions with monomeric ferrous heme followed by difference absorption spectroscopy suggests that some inclusion body VHb is competent for heme binding. A series of perturbation-response experiments was conducted to determine what cellular processes influence VHb IB formation. Results show that VHb inclusion body formation is highly influenced by the expression plasmid construction and the heme biosynthetic capacity. The level of induction and accumulation appear less important than the general metabolic state of the culture. Temperature and chaperone protein levels have little effect. Efforts to reduce inclusion body formation through genetic amplification of ALA synthase and ALA dehydratase levels were unsuccessful, presumably due to regulation. Results suggest a heme biosynthetic limitation is involved in VHb in vivo insolubilization.

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Huang, Ji-Tzeng, i 黃繼增. "Refolding of the recombinant protein that forms inclusion bodies in Escherichia coli". Thesis, 2000. http://ndltd.ncl.edu.tw/handle/13919387326374696082.

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碩士
國立中興大學
農業生物科技學研究所
88
Abstract The Escherichia coli expression system is by far the best way for the over-expression of recombinant proteins. However, one of the major obstacles in utilizing E. coli for over-expressing recombinant proteins arises from inclusion body formation. The over-expressed recombinant proteins appear in an insoluble form. In order to make the recombinant proteins soluble, strong protein denaturant such as 8 M urea or 6 M guanidinium hydrochloride is used. Subsequently, urea or guanidinium hydrochloride is removed gradually by dialysis to allow refolding of the denatured proteins. In this study, by making use of the specific binding between 6×His tag and Ni2+, I explored the possibility of efficient protein refolding upon Ni-affinity chromatography. A chimeric gene transcribed from a T7 promoter for over-expressing a 6×His-tagged TrxA-GST3 fusion protein was constructed. By following the specific activities of thioredoxin and glutathione S-transferase, I could determine and compare the refolding efficiencies quantitatively. Analysis of the results indicated that, either by dialysis or upon Ni-affinity chromatography, the refolding efficiency of the thioredoxin activity could reach as high as approximately 50%. This result suggests that it is possible to achieve the refolding efficiency achievable by dialysis by refolding upon Ni-affinity chromatography. In contrast, while the refolding efficiency of the glutathione S-transferase activity by dialysis could reach 56% (at the protein concentration of 0.075 mg/ml), the refolding in Ni-affinity chromatography was only one-sixth as efficient. Moreover, the recovery of glutathione S-transferase activity by dialysis was apparently influenced by protein concentration. At the protein concentrations of 0.075-0.185 mg/ml, the refolding efficiencies could reach 40-56 %. When the protein concentration was above or below 0.075-0.185 mg/ml, the refolding efficiencies dropped to 15-25 %. On the other hand, the recovery of thioredoxin activity by dialysis was relatively unaffected by protein concentration.

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Huang, Shin-Wei, i 黃信維. "Minimize Periplasmic Inclusion Body Formation for Overproduction of Recombinant Penicillin Acylase in Escherichia coli". Thesis, 2001. http://ndltd.ncl.edu.tw/handle/25214746240080982345.

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碩士
逢甲大學
化學工程學系
89
To produce penicillin acylase (PAC) by recombinant DNA technology in Escherichia coli (E. coli), the overproduction was often limited by periplasmic processing and inclusion bodies were formed at a large amount in the periplasm. This raises an important issue that, for the overproduction of recombinant proteins, not only the transcriptional and/or translational efficiency has to be increased but also a ‘balanced’ protein synthesis flux throughout various gene expression (i.e., transcription, translation, and posttranslational processing) and folding steps should be properly maintained to avoid the accumulation of polypeptide intermediates. In this study, we demonstrated the extracellular production of penicillin acylase (PAC) by coexpression of the brp gene encoding bacteriocin release protein (BRP) and the pac gene. The performance for the production and release of PAC was optimized by taking several culture parameters, including host, inducer concentration, and induction timing for brp expression, into consideration. The effect of PAC release on inclusion body formation was also investigated. It was observed that the amount of inclusion bodies was significantly increased by brp expression. The formation of inclusion bodies was not caused by over-accumulation of active PAC. In the second part of this study, we demonstrated the enhancement of recombinant penicillin acylase (PAC) production in E. coli by increasing the intracellular concentration of the periplasmic protease DegP. The amount of these periplasmic inclusion bodies was significantly reduced and PAC activity was significantly increased upon coexpression of DegP. The results suggest that DegP could in vivo assist the periplasmic processing though the enzyme was shown to be not absolutely required for the formation of active PAC in E. coli. The formation of PAC inclusion bodies should be primarily caused by limitation of the proteolysis on periplasmic PAC precursors. In addition, the steps limiting the production of PAC were identified.

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CHENG, CHIH-YU, i 鄭志宇. "Purification of Recombinant AsChi61, a chitinase in Aeromonas schubertii from Inclusion Body of Escherichia coli". Thesis, 2017. http://ndltd.ncl.edu.tw/handle/v8rpf3.

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Li, Ruei-Yu, i 李瑞俞. "Study of Inclusion Body Formation under Various Culture Conditions in Recombinant Escherichia coli with Bioimaging System". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/01602861630330623536.

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碩士
國立成功大學
化學工程學系碩博士班
95
The bioimaging system usually utilizes fluorescence protein as a reporter gene in eukaryotic systems, and it gives us an easy way to real-time monitor the distribution of fluorescence protein in cells. Eukaryote is thousand times the size of prokaryote, so it is not suitable to this system. In our research, giant protoplasts with size similar to Saccharomycete were prepared from recombinant Escherichia coli BL21(DE3)/pET-D7. The expression of D7 can be induced by IPTG to monitor inclusion body formation in real time. Using this approach, we can study inclusion body formation in recombinant Escherichia coli under various culture conditions. By the fluorescence microscope, we monitored the expression of fluorescence in the giant protoplast. The difference in fluorescence under various culture contitions revealed that inclusion body formation would increase with increasing IPTG, with increasing induction temperature or with decreasing pH value.

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26

JunXie, Yao, i 謝曜駿. "Study of Green Fluorescence Protein Inclusion Body Formation under Various Culture Conditions in Recombinant Escherichia coli with Bioimaging System". Thesis, 2010. http://ndltd.ncl.edu.tw/handle/39510713938155070497.

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碩士
國立成功大學
化學工程學系碩博士班
98
Bioimaging system usually utilize fluorescence protein as a reporter gene in eukaryotic system, and it gives a easy way for us to real-time monitor the distribution of fluorescence protein in bacteria or cell.Eukaryote is thousand times the size of prokaryote, so it is not suitable to this system.In our reserach, recombinant Escherichia coli BL21(DE3)/ pET21a-GFP is used to prepare as giant protoplast which size is similar to saccharomycete with particular method. The expression of GFP can be induced by IPTG to real-time monitor inclusion body formation.Using this approach, we can study inclusion body formation under various culture conditions in recombinant Escherichia coli. By monitoring the fluorescence protein formation in the giant protoplast under various culture conditions, the result indicated that inclusion body formation would increase as increasing IPTG, increasing induction temperature or deceeasing ph value.

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27

Narayanan, Niju. "Molecular and Genetic Strategies to Enhance Functional Expression of Recombinant Protein in Escherichia coli". Thesis, 2009. http://hdl.handle.net/10012/4721.

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The versatile Escherichia coli facilitates protein expression with relative simplicity, high cell density on inexpensive substrates, well known genetics, variety of expression vectors, mutant strains, co-overexpression technology, extracytoplasmic secretion systems, and recombinant protein fusion partners. Although, the protocol is rather simple for soluble proteins, heterologous protein expression is frequently encountered by major technical limitations including inefficient translation, formation of insoluble inclusion bodies, lack of posttranslational modification mechanisms, degradation by host proteases, and impaired cell physiology due to host/protein toxicity, in achieving functional expression of stable, soluble, and bioactive protein.. In this thesis, model protein expression systems are used to address the technical issues for enhancing recombinant protein expression in E. coli. When yellow fluorescence protein (YFP) was displayed on E. coli cell surface, the integrity of the cell envelope was compromised and cell physiology was severely impaired, resulting in poor display performance, which was restored by the coexpression of Skp, a periplasmic chaperone. On the basis of monitoring the promoter activities of degP, rpoH, and cpxP under various culture conditions, it was demonstrated that the cell-surface display induced the σE extracytoplasmic stress response, and PdegP::lacZ was proposed to be a suitable “sensor” for monitoring extracytoplasmic stress. Intracellular proteolysis has been recognized as one of the key factors limiting recombinant protein production, particularly for eukaryotic proteins heterologously expressed in the prokaryotic expression systems of E. coli. Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression. The study offers an alternative genetic strategy in molecular manipulation to enhance recombinant protein production in E. coli. To overcome the technical limitations of protein misfolding, ineffective disulfide bond formation, and protein instability associated with intracellular proteolysis in the functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli, an alternative approach was explored by extracellular secretion of PalB via two Sec-independent secretion systems, i.e. the α-hemolysin (Type I) and the modified flagellar (Type III) secretion systems, which can export proteins of interest from the cytoplasm directly to the exterior of the cell. Bioactive PalB was expressed and secreted extracellularly either as HlyA fusion (i.e. PalB-HlyA via Type I system) or an intact protein (via Type III system) with minimum impact on cell physiology. However, the secretion intermediates in the intracellular fraction of culture samples were non-bioactive even though they were soluble, suggesting that the extracellular secretion did mediate the development of PalB activity. PalB secretion via Type I system was fast with higher specific PalB activities but poor cell growth. On the other hand, the secretion via Type III system was slow with lower specific PalB activities but effective cell growth. Functional expression of lipase from Burkholderia sp. C20 (Lip) in various cellular compartments of Escherichia coli was explored. The poor expression in the cytoplasm was improved by several strategies, including coexpression of the cytoplasmic chaperone GroEL/ES, using a mutant E. coli host strain with an oxidative cytoplasm, and protein fusion technology. Fusing Lip with the N-terminal peptide tags of T7PK, DsbA, and DsbC was effective in boosting the solubility and biological activity. Non-fused Lip or Lip fusions heterologously expressed in the periplasm formed insoluble aggregates with a minimum activity. Biologically active and intact Lip was obtained upon the secretion into the extracellular medium using the native signal peptide and the expression performance was further improved by coexpression of the periplasmic chaperon Skp. The extracellular expression was even more effective when Lip was secreted as a Lip-HlyA fusion via the α-hemolysin transporter. Finally, Lip could be functionally displayed on the E. coli cell surface when fused with the carrier EstA.

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Vallejo, Gonzalez Luis Felipe. "Technical and kinetic aspects of the in vitro refolding process of bone morphogenetic protein-2 from Escherichia coli produced inclusion bodies /". 2006. http://www.gbv.de/dms/bs/toc/517553430.pdf.

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Le, Thanh Ha [Verfasser]. "Optimisation of active recombinant protein production, exploring the impact of small heat-shock proteins of Escherichia coli, IbpA and IbpB, on in vivo reactivation of inclusion bodies / by Than Ha Le". 2005. http://d-nb.info/975691554/34.

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Russell, Bonnie Leigh. "Expression, solubilisation, purification and characterisation of recombinant bluetongue virus viral protein 7". Diss., 2018. http://hdl.handle.net/10500/24951.

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Bluetongue virus belongs to the Orbivirus genus from the Reoviridae family. It infects predominantly domestic and wild ruminants and is economically significant worldwide. Bluetongue virus VP7 forms the intercepting layer between the outer capsid (VP2 and VP5) and VP3 which surrounds the genomic material. BL21(DE3), NiCo21(DE3), C43(DE3) pLysS and KRX Escherichia coli cells were transformed with a pET28a plasmid with the cDNA sequence encoding Bluetongue virus VP7. Expression of Bluetongue virus VP7 was tested at post induction temperatures between 16˚C and 37 ˚C, at inducer concentrations between 0.1 mM and 1.0 mM isopropyl-β-D-thiogalactopyranoside in BL21(DE3), NiCo21(DE3) and C43(DE3) pLysS cells and 0.05 % and 0.15 % rhamnose for KRX cells, in two types of growth media (LB and 2xYT) and post-induction growth times between two and 16 hours. Under all conditions tested; Bluetongue virus VP7 expression was found to be predominantly in the insoluble fraction (pellet). BL21(DE3) and NiCo21(DE3) cells were chosen and grown for five hours post induction, induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside and grown at a post-induction temperature of 37 ˚C. Bluetongue virus VP7 in bacterial cell inclusion bodies was solubilised using urea and a freeze-thaw step. Solubilisation was tested with urea concentrations between 2 M and 8 M, with solubilisation efficiency not increasing past 5 M urea. Solubilized Bluetongue virus VP7 was purified using nickel-affinity chromatography. Purified Bluetongue virus VP7 was then probed with far-UV circular dichroism and intrinsic fluorescence in several buffer conditions including different urea and guanidinium chloride concentrations as well as in the presence of glycerol and sodium chloride. Guanidinium chloride was able to cause Bluetongue virus VP7 unfolding, and the unfolding transition had 94 % and 89 % reversibility at 218 nm and 222 nm respectively. Bluetongue virus VP7 was shown to contain a native-like structure in 20 % glycerol and in up to 8 M urea and was found to be stable till at least 55 ˚C, even in the presence of 5 M urea. Glycerol and sodium chloride influenced the conformation of the protein resulting in different unfolding transitions. Thermal unfolding of Bluetongue virus VP7 was found to be irreversible.
Life and Consumer Sciences
M. Sc. (Life Sciences)

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