Clonagem, expressão e purificação de membros da família de Proteína de Superfície Associada a Mucina (MASP) de Trypanosoma cruzi para estudos estruturais

• Ano de defesa: 2010
• Autor(a) principal: Simara Semíramis de Araújo
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: Proteína de Superfície Associada a Mucina; Cristalografia de proteínas; Trypanosoma cruzi; Bioquimica Tecnica; Tripanossoma cruzi; Proteína de superfície associada a mucina; Proteinas Estrutura
• Link de acesso: http://hdl.handle.net/1843/BUOS-97PGB9

The Trypanosoma cruzi masp (mucin associated surface protein) gene family was identified during the annotation of the genome as the second largest gene family in this human pathogen, consisting of approximately 1400 members. MASP members contain N- and C- terminal conserved domains that encode a putative signal peptide and a GPI-anchor addition site, respectively. The central region is variable both in length and in amino acid sequence and contains a large repertoire of repetitive motifs, which suggests MASP may be involved in parasite-host cell interaction. Since no member of the MASP family has been characterized to date, we have decided to study the secondary and tertiary structures of distinct MASPs. For this purpose, we have decided to clone and express in E. coli the central region sequences of four MASP members. For this, some features of the target proteins were considered such as short amino acid sequence length, around 170 residues, low content of potential glycosylation sites and low intrinsic disorder degree. Knowing that sequence regions with low complexity nearly always correspond to nonfolding segments or extended structures, we have used the web server PONDR VL-XT to predict disordered regions in MASPs. Natively unfolded proteins have been described to possess a high ratio of charged residues and a low ratio of hydrophobic residues. The mean net charge versus mean hydrophobicity ratio was ploted for the four MASPs central region. This analysis showed that the four central regions falls in the phase space of natively unfolded proteins. After that, the sequences were PCR amplified and cloned into TOPO and/or pGEM-T cloning systems and submitted to DNA sequencing. Subsequently, the PCR products were transferred to two expression vectors derived from pET-28a vector (Novagen), which were modified in the Center of Molecular and Structural Biology (CeBiME, Campinas/SP). In both vectors, the thrombin recognition site was replaced by a TEV (tobacco etch virus) protease recognition site. The four N-terminal fusion His-tag MASP proteins were expressed in the soluble forms, whereas the N-terminal GST fusion MASPs were expressed as insoluble inclusion bodies, except the recombinant GST:MASP1. Purification of the Histidine-tagged MASP3 was carried out by affinity chromatography. After His-tag cleavage by TEV, the protein was purified by ion exchange chromatography. The study of the three-dimensional structure of MASP proteins will enable a more detailed understanding of these molecules, such as the location of their epitopes and its fold, which may contribute to the identification of its biological function on T. cruzi.