Publicação
Production and Characterization of stimuli-responsive marine proteins
| Resumo: | Cephalopods, a family of marine invertebrates, have been a source of inspiration in the scientific community for their ability to modulate coloration and texture. The production of structural coloration is associated with reflectins, a family of proteins that exist in skin organs and that act as Bragg reflectors. Since their discovery, reflectins have been explored for the production of stimuli-responsive bio-based materials. This thesis aimed at the recombinant production, purification and characterization of reflectin proteins. Two reflectins were selected from different organisms – Reflectin 1 and Reflectin 2. Both proteins were expressed in Escherichia coli in a rich culture medium and the proteins were then purified by Immobilized Metal Affinity Chromatography method. The purification of reflectin 2 resulted in a lower recovery yield (7%) in comparison to reflectin 1(27%). Thus, the purification of reflectin 2 was attempted by Reverse-Phase Chromatography resulting in a higher yield (41%). Reflectin 1 was purified with a final of 73%, while reflectin 2 was purified by both chromatographic methods with a final of 95%. Biophysical properties of the proteins were characterized by several techniques such as Circular Dichroism to determine the secondary structure and to evaluate the thermal stability of the proteins; and Dynamic Light Scattering to determine the particle size and charge in different pH values (1.5, 2.0, 3.0, 8.0 and 11.1). The proteins were characterized as intrinsically disordered proteins as no predominant secondary structure was observed. Protein’s self-assembly property is influenced by a progressive neutralization of the charge. The reversible self-assembly is related to the number of histidines in the reflectin sequence, reflectin 2 demonstrated the ability to reversible the self-assembly upon a cyclical pH change. Overall, the thesis reports for the first time on the production of reflectin 2 and the comparison of structural characterization between reflectin 1 and reflectin 2. |
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| Autores principais: | Soares, Cátia Lopes |
| Assunto: | Reflectins Self-Assembly Reversible Self-Assembly |
| Ano: | 2021 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade Nova de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório Institucional da UNL |
| Resumo: | Cephalopods, a family of marine invertebrates, have been a source of inspiration in the scientific community for their ability to modulate coloration and texture. The production of structural coloration is associated with reflectins, a family of proteins that exist in skin organs and that act as Bragg reflectors. Since their discovery, reflectins have been explored for the production of stimuli-responsive bio-based materials. This thesis aimed at the recombinant production, purification and characterization of reflectin proteins. Two reflectins were selected from different organisms – Reflectin 1 and Reflectin 2. Both proteins were expressed in Escherichia coli in a rich culture medium and the proteins were then purified by Immobilized Metal Affinity Chromatography method. The purification of reflectin 2 resulted in a lower recovery yield (7%) in comparison to reflectin 1(27%). Thus, the purification of reflectin 2 was attempted by Reverse-Phase Chromatography resulting in a higher yield (41%). Reflectin 1 was purified with a final of 73%, while reflectin 2 was purified by both chromatographic methods with a final of 95%. Biophysical properties of the proteins were characterized by several techniques such as Circular Dichroism to determine the secondary structure and to evaluate the thermal stability of the proteins; and Dynamic Light Scattering to determine the particle size and charge in different pH values (1.5, 2.0, 3.0, 8.0 and 11.1). The proteins were characterized as intrinsically disordered proteins as no predominant secondary structure was observed. Protein’s self-assembly property is influenced by a progressive neutralization of the charge. The reversible self-assembly is related to the number of histidines in the reflectin sequence, reflectin 2 demonstrated the ability to reversible the self-assembly upon a cyclical pH change. Overall, the thesis reports for the first time on the production of reflectin 2 and the comparison of structural characterization between reflectin 1 and reflectin 2. |
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