<p>A method describing the expression and purification of PSI-N together with fusion protein, using affinity chromatography and TEV protease. Although the method proved successful, optimization is still needed due to partial degradation of PSI-N.</p>

A method describing the expression and purification of PSI-N together with fusion protein, using affinity chromatography and TEV protease. Although the method proved successful, optimization is still needed due to partial degradation of PSI-N.

Grouper (Epinephelus coioides) is one of the important farmed fish in the southern Taiwan. However, grouper aquaculture in Taiwan has a serious problem of infection, especially in grouper larvae breeding stage. The infection resulted in very high mortality, which causes massive economic loss. Therefore, early detecting the presence of pathogen is critical for preventing epidemic outbreak. Interleukin-1 (IL-1) is one of proinflammatory cytokines that form a feedback control loop with anti-inflammatory cytokines to maintain the homeostasis of host immune response. The increase of IL-1 expression could be an indicator of pathogenic insult. In this study, total RNA of Epinephelus coioides fertilized egg was extracted for reverse transcription-polymerase chain reaction (RT-PCR) to amplify cDNA of IL -1£]. The cDNA amplified was then cloned into pGEX4T-3 for the expression and purification of GST-IL-1£] fusion protein. GST-IL-1£] fusion protein purified was then used to immunize New Zealand white rabbit for generation of antiserum against IL-1£]. Western blot result confirmed the specificity of antiserum as the immune serum, but not the preimmune serum, detected the immunogen GST-IL-1s. Further experiments using live Epinephelus coioides injected with or without lipopolysarcharides (LPS) further confirmed that this antiserum could detect a massive increase of IL-1£] protein after the injection of LPS in either protein lysate by western blotting or in frozen tissue section of head kidney by immunohistochemistry. In summary, we successfully generated a rabbit specific antiserum against IL-1£] of Epinephelus coioides , which could be a useful reagent for future analysis of fish immune response upon pathogen infection.

Small fibrils termed actinotrichia are involved with the growth and structure of the fin fold during fin development in fish. The actinodin (and) genes are required for actinotrichia formation, and the loss of these genes from the genomes of tetrapods has been implicated in the tetrapod-specific loss of actinotrichia, loss of a fin fold and the concurrent evolution of paired fins into limbs. This study focuses on the function of the and genes and their role in actinotrichia formation. The results reveal cis-acting regulatory elements required for and1 expression in the fin epithelium. Furthermore, it is shown that the And proteins display similarities to the secreted signaling molecule, Ecrg4, implying a possible role in cell differentiation during fin fold development. In the final section of this report, I use a genomic analysis to show that the and genes were lost from otherwise well-conserved syntenic loci in fish and tetrapod genomes. These results suggest possible causes for the evolutionary loss of and genes and the associated developmental changes that may have permitted fin to limb evolution.

Ferrochelatase (FeCH) is an ubiquitous enzyme producing heme, an essential pigment for all forms of organisms. In photosynthetic organism, heme is synthesized together with the chlorophyll in one branched pathway and the FeCH enzyme appears to be important for regulation of both the chlorophyll and the heme biosynthesis. To understand regulatory role of this protein, an active recombinant FeCH from photosynthetic organism would be invaluable. The aim of this project is to express FeCH from cyanobacterium Synechocystis 6803 in Escherichia coli and to prepare a protocol for the purification of this protein as a highly active enzyme.

Orientador: Fátima Pereira de Souza / Banca: Karina Alves de Toledo / Banca: José Roberto Ruggiero / Resumo: O Vírus Sincicial Respiratório Humano (hRSV) foi identificado em 1957 e mesmo após vários anos de investigação, nenhuma vacina foi desenvolvida. Acredita-se que a chave de inibição da ação viral são suas glicoproteínas de membrana, em especial a proteína de fusão (F), que com auxílio da proteína de ligação (G), é responsável pela instalação do hRSV na célula hospedeira. Há evidências experimentais de que compostos como flavonóides e glicosaminoglicanos podem diminuir a infecção viral, sendo então a proteína F um bom alvo para a ação destes compostos. O presente estudo utilizou de ferramentas de bioinformática para verificar as possíveis regiões de interação da proteína F com a Heparina Sulfatada e Flavonóides. Os programas de bioinformática foram utilizados para: modelagem dos compostos, caracterização e previsão da estrutura secundária da proteína, modelagem da estrutura terciária e docking molecular entre o modelo da proteína F e as estruturas tridimensionais dos Flavonóides e da Heparina Sulfatada. Modelos válidos foram obtidos para as estruturas tridimensionais dos flavonóides e para o modelo completo da proteína F. As características da proteína incluem um alto nível de conservação na seqüência de aminoácidos e, especialmente, em seus sítios de ligação. O docking da proteína com a Heparina, e o virtual screening da biblioteca de Flavonóides e a estrutura da proteína, resultaram em sítios de interação com grande potencial de inibição, uma vez que concordam com evidências experimentais descritos na literatura. A Heparina liga-se ao sítio de clivagem II, importante região para obtenção da atividade de fusão da proteína. Os Flavonóides podem se ligar a região hidrofóbica que desestabiliza... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Human Respiratory Syncytial Virus (hRSV) was identified in 1957 and even after several years of research, no vaccine has been developed yet. It is believed that the key to the inhibition of viral action is its membrane glycoproteins, including the Fusion Protein (F), responsible for the installation of the hRSV in the host cell. There are evidences that compounds such as flavonoids and glycosaminoglycans can decrease the viral infection, and F protein can be a good target for the action of these compounds. The present study checked the possible sites of interaction between F protein and heparin and flavonoids, using computational tools. Bioinformatics programs were used for: modeling compounds, characterization and prediction of protein secondary structure, tertiary structure modeling and the docking between the protein model and the structures of flavonoids and sulfated heparin. Valid models were obtained for flavonoids structures and the complete model of F protein. The characteristics of the protein include a high level of conservation in amino acid sequence and especially in its binding sites. The heparin docking and virtual screening of flavonoids resulted in interaction sites with great potential for inhibition, since they agree with other studies and experimental evidence of F protein inhibition. This study shows that compounds such as sulfated heparin and flavonoids interact in important sites of F protein. Heparin binds to the cleavage site II and flavonoids can bind to the hydrophobic site that destabilizes the formation of the six-helix-bundle region. Both regions are important for conformational changes that F protein undergoes to get its fusion activity. Docking showed that molecular interactions are likely to occur and selected the best candidates for a possible inhibitor. These evidences... (Complete abstract click electronic access below) / Mestre

There is increasing concern regarding alternative, sustainable energy sources, such as biofuels, to replace declining oil reserves. The abundance of lignocellulosic biomass makes it the only imaginable resource that can potentially substitute a substantial portion of the fossil fuels we use today, but current methods for producing biofuels from non-food crops are cost intensive and not economically viable. Synthetic biology provides several potential approaches for developing biologically mediated processes for the conversion of lignocellulosic biomass into biofuels. Such systems are based on engineered microbes that produce enzymes for catalysing the conversion of cellulose into fermentable sugars and subsequently into high value products. Effective degradation of cellulose requires multiple classes of enzyme working together. In naturally occurring cellulose degrading microbes, bioconversion is catalysed by a battery of enzymes with different catalytic properties. However, naturally occurring cellulases with multiple catalytic domains seem to be rather rare in known cellulose-degrading organisms. Using synthetic biology approaches, seven cellulases with multiple catalytic domains were engineered and tested to determine the usefulness of such chimeric enzymes to replace cloning of multiple enzymes for biomass conversion. Catalytic domains were taken from Cellulomonas fimi endoglucanases CenA, CenB and CenD, exoglucanase Cex, and β-glucosidase, Cfbglu as well as Cytophaga hutchinsonii cellodextrinase CHU2268. All fusions retained both catalytic activities of the parental enzymes. To investigate the benefits of fusion, Citrobacter freundii NCIMB11490 was transformed with either fused or non-fused enzymes and cultured with cellulose blotting papers as main carbon source. Cells expressing fusions of Cex with CenA or CenD reproducibly showed higher growth than cells expressing non-fused versions, as well as more rapid physical destruction of paper. The opposite was observed for the other combinations. Comparing two different Cex and CenA fusions, CxnA2, which contains two carbohydrate binding modules (CBMs), degraded filter paper faster and led to better growth than CxnA1, which contains only one CBM. It was observed that CxnA1 was exported to the supernatant of E. coli and C. freundii cultures, as also seen for Cex and CenA, although there is no clear biological mechanism for this. Monitoring of growth using colony counts is laborious, but the use of optical density is not possible for cellulose-based cultures as it is affected by the insoluble cellulose particles. The SYBR Green I/propidium iodide live/dead staining protocol was therefore evaluated for growth measurements and was found to allow rapid measurements of large numbers of samples. In conclusion, these studies have demonstrated a simple and useful method for making chimeric proteins from libraries of multiple parts. The results demonstrate that use of fusion proteins can improve biomass conversion in vivo, and could potentially reduce the necessity for cloning of multiple enzymes and improve product yields. A simple and effective method for monitoring growth of bacteria in turbid cultures using a fluorimeter has also been developed.

Small fibrils termed actinotrichia are involved with the growth and structure of the fin fold during fin development in fish. The actinodin (and) genes are required for actinotrichia formation, and the loss of these genes from the genomes of tetrapods has been implicated in the tetrapod-specific loss of actinotrichia, loss of a fin fold and the concurrent evolution of paired fins into limbs. This study focuses on the function of the and genes and their role in actinotrichia formation. The results reveal cis-acting regulatory elements required for and1 expression in the fin epithelium. Furthermore, it is shown that the And proteins display similarities to the secreted signaling molecule, Ecrg4, implying a possible role in cell differentiation during fin fold development. In the final section of this report, I use a genomic analysis to show that the and genes were lost from otherwise well-conserved syntenic loci in fish and tetrapod genomes. These results suggest possible causes for the evolutionary loss of and genes and the associated developmental changes that may have permitted fin to limb evolution.

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Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.