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Cloning, expression and purification of a sea urchin adhesive protein towards the development of a new biomedical adhesive
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Os ouriços do mar vivem em zonas de elevado hidrodinamismo, e para sobreviverem dispõe de órgãos adesivos, os pés ambulacrários, especializados na locomoção e adesão ao substrato. Embora se fixem fortemente ao substrato, estes órgãos fazem no de forma temporária, colando-se e descolando-se repetidamente. Os pés ambulacrários orais estão extraordinariamente bem adaptados à adesão temporária, sendo constituídos por um disco apical achatado, com o qual se fixam ao substrato, e um caule extensível que liga o disco à carapaça do ouriço. O disco é o local de produção da secreção adesiva, que fixa o ouriço ao substrato, bem como da secreção “desadesiva” com o qual se descola do mesmo. Até aos anos 90, foi demonstrado que a adesão temporária dos ouriços resulta da presença de um sistema duo-glandular na epiderme do disco, composto por dois ou mais tipos de células que produzem separadamente secreções adesivas e “desadesivas”. A secreção adesiva permanece
fixa ao substrato sob a forma de pequenos círculos (material adesivo secretado) enquanto a secreção “desadesiva” não fica incorporada nestes círculos, atuando provavelmente como um enzima. Foi demonstrado recentemente que o disco dos pés ambulacrários dos ouriços do mar adere com tenacidades (força adesiva/ unidade de área) semelhantes às de outros adesivos marinhos e sintéticos, sendo eficaz em substratos com diferentes rugosidades e propriedades químicas. Estes resultados reforçam o potencial biotecnológico do adesivo secretado pelos ouriços do mar, uma vez que, dada a sua resistência, eficácia e versatilidade em meios aquosos e em vários tipos de substratos, poderá ter aplicações industriais e biomédicas. Apesar destas promissoras aplicações, pouco se conhece sobre os mecanismos moleculares responsáveis pela adesão temporária dos ouriços do mar. A caracterização bioquímica do adesivo secretário pelo ouriço do mar Paracentrotus lividus (Pl) e do proteoma dos discos adesivos dos pés ambulacrários da mesma espécie foi recentemente realizada e várias proteínas que parecem estar envolvidas na sua adesão temporária foram identificadas. Neste trabalho, descrevo a purificação da Pl Nectin, uma das possíveis proteínas candidatas à existência de adesão. Para tal, o gene da Pl Nectin foi clonado e sequenciado usando primers desenhados com base nas sequências peptídicas obtidas previamente. O cDNA correspondente foi clonado em plasmídeos e expresso em bactéria. Por fim, a purificação da proteína por cromatografia de afinidade foi tentada, sem sucesso no entanto, apesar de todos os nossos esforços.
Sea urchins inhabit wave-swept shores being subjected to substantial hydrodynamic forces. They thus, rely on adhesive organs, the adoral tube feet, specialized in locomotion and anchoring. These adhesive appendages attach strongly but reversibly to the substratum, detaching and re-attaching voluntarily. Adoral tube feet are well designed for temporary adhesion, possessing an enlarged and flattened apical disc, with which they attach and detach from the substratum, connected to the animal body by an extensible tether, the stem. The tube foot disc produces the adhesive secretion that fastens the animal to the substratum and the de-adhesive secretion that allows it to move. Before the 90s it was only known that the temporary nature of sea urchin adhesion is due to the presence of a duogland adhesive system in the disc epidermis which comprises 2 or more cell types capable of producing separately adhesive and de-adhesive secretions. After detachment, the adhesive secretion remains on the substratum as a footprint (circle of secreted adhesive). As for the de-adhesive secretion it is not incorporated in the footprint, indicating that it might act as an enzyme. It was recently shown that the tube feet discs attach with similar tenacity (adhesive force per unit area) to other marine and commercial adhesives, being effective on several substrata with variable chemistries and roughness. These results reinforce the biotechnological potential of the adhesive secreted by sea urchins, because given its resistance, effectiveness and versatility on aqueous media and several types of substrata, it may find both industrial and biomedical applications. However, despite these promising applications, little is known on the molecular mechanisms behind tube foot temporary adhesion. The biochemical characterization of the adhesive secreted by the sea urchin Paracentrotus lividus (Pl) and the proteome of the tube foot adhesive discs of the same species was recently done and several proteins that seem to be involved in the temporary adhesion were pin-pointed. Here I describe the purification of the Pl Nectin, one of the possible candidates for this adhesion. To achieve this goal the Pl Nectin gene was cloned and sequenced using primers designed based on the previously obtained peptide sequences. The correspondent cDNA was cloned in plasmids and expressed in bacteria. Finally, the purification of the protein by affinity-chromatography was attempted but failed despite all of out tries.
Sea urchins inhabit wave-swept shores being subjected to substantial hydrodynamic forces. They thus, rely on adhesive organs, the adoral tube feet, specialized in locomotion and anchoring. These adhesive appendages attach strongly but reversibly to the substratum, detaching and re-attaching voluntarily. Adoral tube feet are well designed for temporary adhesion, possessing an enlarged and flattened apical disc, with which they attach and detach from the substratum, connected to the animal body by an extensible tether, the stem. The tube foot disc produces the adhesive secretion that fastens the animal to the substratum and the de-adhesive secretion that allows it to move. Before the 90s it was only known that the temporary nature of sea urchin adhesion is due to the presence of a duogland adhesive system in the disc epidermis which comprises 2 or more cell types capable of producing separately adhesive and de-adhesive secretions. After detachment, the adhesive secretion remains on the substratum as a footprint (circle of secreted adhesive). As for the de-adhesive secretion it is not incorporated in the footprint, indicating that it might act as an enzyme. It was recently shown that the tube feet discs attach with similar tenacity (adhesive force per unit area) to other marine and commercial adhesives, being effective on several substrata with variable chemistries and roughness. These results reinforce the biotechnological potential of the adhesive secreted by sea urchins, because given its resistance, effectiveness and versatility on aqueous media and several types of substrata, it may find both industrial and biomedical applications. However, despite these promising applications, little is known on the molecular mechanisms behind tube foot temporary adhesion. The biochemical characterization of the adhesive secreted by the sea urchin Paracentrotus lividus (Pl) and the proteome of the tube foot adhesive discs of the same species was recently done and several proteins that seem to be involved in the temporary adhesion were pin-pointed. Here I describe the purification of the Pl Nectin, one of the possible candidates for this adhesion. To achieve this goal the Pl Nectin gene was cloned and sequenced using primers designed based on the previously obtained peptide sequences. The correspondent cDNA was cloned in plasmids and expressed in bacteria. Finally, the purification of the protein by affinity-chromatography was attempted but failed despite all of out tries.
Descrição
Tese de mestrado em Bioquímica Médica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015
Palavras-chave
Bioquímica médica Teses de mestrado - 2015