Fome e Abundância: Um Paradoxo Brasileiro?
17 a 22 de outubro de 2016
Trabalho 6738
| ISSN | 2237-9045 |
|---|---|
| Instituição | Universidade Federal de Viçosa |
| Nível | Pós-graduação |
| Modalidade | Pesquisa |
| Área de conhecimento | Ciências Biológicas e da Saúde |
| Área temática | Farmacologia e bioquímica |
| Setor | Departamento de Biologia Geral |
| Bolsa | CAPES |
| Conclusão de bolsa | Não |
| Apoio financeiro | CAPES, CNPq, FAPEMIG |
| Primeiro autor | Christiane Eliza Motta Duarte |
| Orientador | LEANDRO LICURSI DE OLIVEIRA |
| Outros membros | Andréa Dias Koehler, Josefa Muñoz Alamillo |
| Título | Molecular cloning, expression and characterization of a new TRAF-like protein with lectin activity |
| Resumo | Lectins are proteins widely distributed in nature that contain at least one non-catalytic domain that recognize and bind in a reversible way to specific glycans, either free or as part of glycoproteins or glycolipids. Plant lectins are often isolated from their natural sources. Purification, however, could be both laborious and expensive and does not guarantee high protein yield. The advent of recombinant DNA technology made possible the large-scale synthesis and purification of lectins, through the isolation of cDNA or genomic DNA lectin, insertion into appropriate vectors and expression in the host cell. Therefore, heterologous expression, especially in microbial hosts, is a promising tool to improve availability and purification of lectins with activities of interest. BOL is non-glycosylated monomeric lectin extracted from Brassica oleracea ssp. botrytis with potentially exploitable immunomodulatory activity. However, its isolation and purification from cauliflower is time consuming and generally result in low yield. To optimize the process of the lectin obtaining, the current work presents the molecular cloning and heterologous expression of BOL. Degenerated primers were designed from the amino acid sequence of tryptic fragments and N-terminal sequencing from the native protein. Using total RNA extracted from cauliflower seedlings a BOL coding cDNA sequence of 1053 bp was isolated. Analysis of amino acid sequence and alignment with deduced homologous proteins allowed us to determine that the mature protein comprises 301 amino acids. Predicted three-dimensional structure showed that this lectin had an overall dome-like structure with two MATH-domains. BOL is the first lectin identified with MATH-domains and it can show a new mechanism of interaction yet unidentified in other TRAF proteins. The expression of the recombinant lectin in Escherichia coli resulted in a soluble protein with hemagglutination activity and similar properties as the native lectin. The production of active rBOL in good yields opens up the possibility of obtaining its tridimensional structure as well as making feasible subsequent investigations about structure and function of this intriguing TRAF-like protein with lectin activity. |
| Palavras-chave | heterologous expression, lectin L-type, MATH-domain. |
| Forma de apresentação..... | Painel, Oral |