Collagenase for biotechnology applications

• Autor(a) principal: Abreu, Sara Priscila Lopes de
• Data de Publicação: 2015
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/15507
• topic: Biologia molecular e celular; Biotecnologia; Colagénio

Collagen is an abundant protein in all animals and is the main component of numerous tissues, having also structural and regulatory roles. Collagen can be found in various numerous locations presenting specific functions, such as the establishment of the cellular shape, its strength and structural integrity, and is also involved in tissue maintenance. Moreover, collagen has an important role in the formation of the extracellular matrix of the connective tissue, by contributing to its physical properties. Collagenases are enzymes, more specifically metalloproteinases, with a zincbinding domain. These enzymes are able to digest the triple helical region of native collagen, cleaving its peptide bonds. In recent years, bacterial collagenases have been gaining interest for industry proposes, by offering a variety of applications. In the clinical area, collagenases are used as therapy for diseases associated with excessive deposition of collagen, as for example, in patients suffering from Dupuytren’s disease. The enzymes can also be used to help the debridement of wounds and burns, and also for cancer gene therapy applications. The objective of this work is the comparison and optimization of two methods of recombinant collagenase purification, evaluating which method provides the most cost-effective purification. For that purpose, a simple and high resolution method for purification of histidine-tagged recombinant protein was used. Immobilized metal ion affinity chromatography (IMAC) with a Ni Sepharose 6 Fast Flow, through a column HisTrap FF 1ml or in batch were used. Results obtained through protein quantification by densitometry technique, showed that the purification using a column has higher yield. By zimography was possible determine the gelagenolytic activity of ColAh, and was also possible quantify the collagenolytic activity of ColAh, using a synthetic peptide N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGA). Obtained results also showed that the obtained recombinant protein is stable to storage over time and when submitted to different temperatures (room temperature; 4°C; -20°C).