Navegando por Palavras-chave "Crispr"
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- ItemSomente MetadadadosEstudo estrutural da endonuclease cas6 atuante no sistema de defesa imunoadaptativo crispr-cas de procarion(Universidade Federal de São Paulo (UNIFESP), 2016-09-06) Goncalves, Giulliana Augusta Rangel [UNIFESP]; Alfonso, Martin Rodrigo Alejandro Wurtele [UNIFESP]; http://lattes.cnpq.br/0625673643875019; Universidade Federal de São Paulo (UNIFESP)The active protein in mechanisms of post-transcriptional gene silencing have been the target of several studies due to its promising applications in the field of biological sciences by specifically recognizing oligonucleotide sequences target and mediate their cleavage or repression. The Cas6 protein endonuclease belonging to the CRISPR system (regularly interspaced short clustered palindromic repeats) has recently been described in some works by recognizing a specific sequence highly conserved in prokaryotes, first mediating its cleavage and then participating in formation of a complex with other Cas proteins, culminating in the degradation of genetic material attacker by a RNA interference mechanism, conferring immunity through this system archeas and bacteria. Thus, the purpose of this work was the structural study of the Cas6 protein of T.marítima obtained by recombinant DNA technology in E. coli Rosetta (DE3) and cloned, expressed, purified and crystallized. The native and derivative crystals were subjected to tests of diffraction X-rays with resolutions up to 2.2Å being collected various data sets, where they were processed generating statistical data. Based on this data, different methods have been used to solve the protein structure: the method of molecular replacement, isomorphous replacement and anomalous dispersion. In the latter, the incorporation of a heavy metal to the protein during the step of expression was necessary. Despite the adoption of numerous attempts and different strategies, it could not resolve the structure of the endonuclease. However, it was obtained in high concentrations, and is available for future individual tests or in complex with other Cas proteins have been purified by laboratory. The structural knowledge of this protein may contribute substantially to the establishment of inhibitors scan trials and ultimately promote the development of treatmentsfor diseases caused by these organisms.
- ItemSomente MetadadadosExpressão, purificação, cristalização e análise preliminar de dados cristalográficos da proteína csm4 de thermotoga marítima(Universidade Federal de São Paulo (UNIFESP), 2015-11-30) Villegas, Nadia Valeria Gavilan [UNIFESP]; Alfonso, Martin Rodrigo Alejandro Wurtele [UNIFESP]; http://lattes.cnpq.br/0625673643875019; Universidade Federal de São Paulo (UNIFESP)Bacteria and Archaea have developed a defense system against exogenous DNA and RNA, called CRISPR (Clustered Regularly Intespaced Short Palindromic Repeats), which consists of an operon composed of regions of repetitions (repeats) identical to each other, separated by variable regions (spacers) from invading nucleic acids, along with associated proteins (Cas proteins, CRISPR associated proteins) forming an adaptive and heritable immune system. The main objective of this project was to clone, express and purify in recombinant form the Csm4 protein from T. maritima MSB8, and to characterize structurally this protein. This protein is part of a marginally studied complex of Cas ribonucleoproteins termed the Csm complex, which is involved in the targetting of exogenous DNA from bacteriophages and horizontal transfer of nucleic acids. The protein was recombinantly produced in E. coli and purified by affinity chromatography and gel filtration. After concentration, the protein was crystallized in space group P422 with unit cell a=67,5 b=67,5 c=84,8 Å. Data obtained showed that the native crystal diffracts to 2,5 Å at a wavelength of 1,458 Å. In order to solve the structure by MIR other crystals of derivatized protein with platinum and iodide diffracted up to 3,49 and 4,02 Å at wavelengths of 1,06007 and 1,54160 Å, respectively. Additionally, we incorporated selenomethionine into the protein to be able to solve the structure by the MAD (Multi-wavelength Anomalous Diffraction) methodology. These results can serve as a basis for implementing future projects of rational and experimental design of inhibitors in order to prevent the proliferation of these organisms.