Navegando por Palavras-chave "Recombinant Protein"

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    Expressão heteróloga e avaliação in vitro da atividade de proteínas recombinantes de Haementeria vizottoi sobre os mecanismos de coagulação sanguínea, com foco em aplicação biotecnológica
    (Universidade Federal de São Paulo (UNIFESP), 2020-03-05) Linhares, Debora Do Carmo [UNIFESP]; Tavassi, Ana Marisa Chudzinski [UNIFESP]; Universidade Federal de São Paulo
    Salivary secretions from hematophagous animals have been an important source of proteins with biotechnological potential, since there are a number of protein inhibitors that can be isolated and used to modulate the action of enzymes of interest. Many of these inhibitors have evolved to specifically disable key proteases from their hosts, leading to delayed blood coagulation (anticoagulants) and minor immune response, providing the hematophagous with the conditions for successful feeding. Based on transcriptomics of salivary glands of the leech Haementeria vizottoi, were identified sequences of three genes whose predicted proteins have signatures of potential biological activity as protease inhibitors. The aim of this prospective study was to clone, express and characterize these three H. vizottoi derived proteins, namely: Hviz 1276 (hemerythrin-like), Hviz 78 (antistasin-like) and Hviz 340 (cystatinlike). Due to their structural characteristics, antistin and cystatin-like proteins were expressed in P. pastoris (Komagataella pastoris) and hemerythrin-like protein was expressed in E. coli. The proteins of interest were recovered from the supernatant medium or cell extract, and purified by molecular exclusion chromatography and / or affinity chromatography with different resins. To determine their effects on the coagulation cascade, activated thromboplastin time (aPTT) and prothrombin time (PT) were determined using diagnostic kits. Depending on the result, they were also evaluated for the potential to inhibit specific proteases such as FXa, thrombin, elastase, kallikrein, papain and cathepsin L. Hviz 1276 did not demonstrate the expected biological activity and its study was discontinued. The application of partially purified Hviz 78, in the order of 1 µmol/L, was enough to extend the intrinsic-initiated coagulation time by more than 700%. It is still necessary to work on the refinement of purification of this protein and to perform new function tests, since no specific inhibitory activity against any of the proteases tested was identified. The most promising results were obtained with Hviz 340, purified and identified as inhibitor of cysteine proteases papain and cathepsin L, for the latter with ki=7.9 nmol/L. This activity represents new opportunities to evaluate the potential of Hviz 340 as a modulator of cellular activity related to the immune response, possibly anti-inflammatory, since cathepsins are critical for antigen processing and presentation.
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    Uso do modo de operação em contínuo para avaliar aspectos fisiológicos de linhagem recombinante de drosophila melanogaster
    (Universidade Federal de São Paulo (UNIFESP), 2019-02-19) Oliveira, Carla Reis [UNIFESP]; Augusto, Elisabeth De Fatima Pires [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Glycoproteins are complex molecules produced preferentially by animal cells, but their quality pattern rely on the knowledge of cell metabolism and requires engineering tools to ensure greater uniformity and productivity. As long as reports on the metabolism of insect cells are scarce, researches on the physiological aspects of Drosophila*melanogaster are of utmost importance to promote their usage. In order to contribute on the knowledge of this metabolism, the present work aimed to identify growth limiting and inhibitory factors of S2AcRVGP2, a D.*melanogaster S2 cell line stably transfected for the synthesis of Rabies virus glycoprotein (GPV). This study was conducted using TC100 based medium in spinner flasks and in benchtop bioreactor stirred at 100 rpm and with bubbleLfree aeration system. Exploratory cultures on batch mode were carried out with different concentrations of LLcystine (CYS2) and magnesium (Mg2+). The influence on the metabolism of different glucose concentrations (GLC) in the feed medium was studied in a continuous mode. It has been shown that lactate and ammonium did not seem to be growth inhibiting. The supplementation with CYS2 11 mg/L enhanced GPV expression by 75% in batch with initial supplementation and 21% in batch with pulse addition at the end of exponential phase. The evaluation of Mg2+ influence on cell growth kinetic parameters showed that when cultivated with half of its basal concentration, cell production increased 37%, but when cultured on [Mg2+] inferior to 121 mg/L, it limited the growth rate and altered cell membrane morphology. Continuous culture with glucose concentration above 3.75 g/L in the feed medium indicated overflow on glycolysis with consequently limitation of cell growth. The metabolism shifted to a more balanced state when fed with glucose concentration under 2.5 g/L. Cell production increased by 40% and GPV expression doubled in the lowest glucose concentration evaluated (1.25 g/L).