Publicação
A molecular biotechnological approach for the development of silk-elastin-like proteins functionalized with leukemia inhibitory factor
| Resumo: | Recombinant protein-based polymers (rPBPs) are emerging as a new class of biomaterials due to their unique chemical, physical and biological properties. With the development of protein engineering and biotechnology, we are now able to use amino acids to design and produce genetically engineered rPBPs with precise control over sequence and length. Indeed, recombinant DNA technology allows the design and biosynthesis of multifunctional complex molecules with exquisite control over structure, making it possible to combine in the same polypeptide chain the properties of two or more different proteins. This opens an unprecedented possibility to create novel compositions for specific applications through the manipulation of the genetic sequence. The silk-elastin-like proteins (SELPs) are rPBPs composed of silk- and elastinlike blocks that combine in the same polypeptide chain the properties of silk and elastin, two remarkable natural proteins. The silk-like unit imparts thermal and chemical stability, whereas the elastin-like unit is a flexible component. This endows SELPs with unique properties that can be explored as base material for the production of advanced functional materials. Leukemia inhibitory factor LIF is a pleiotropic cytokine playing several roles in the regulation of differentiation, cell growth and inflammation, and is an important cytokine for normal keratinocyte growth and wound healing. The functionalization of SELPs can therefore be exploited to create a new generation of functional materials with high potential of application in the biomedical field. The present work was performed in the Department of Biology in University of Minho in the scope of the Master Course in Molecular Genetics. By recurring to recombinant DNA technology techniques, murine recombinant LIF was used to functionalize a SELP copolymer. For this, two constructions were performed considering the fusion of LIF to the N- and C-terminal of the SELP chain. The recombinant functionalized SELPs were successfully expressed in Escherichia coli and purified by a simple and non-chromatographic method with high recovery rates. Furthermore, aiming at its subsequent use in drug delivery systems, production and purification of sole LIF was also considered. The recombinant proteins were assessed for their bioactivity in C2C12 cells showing promising results and suggesting that these recombinant proteins could have a high potential of application in the biomedical field. |
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| Autores principais: | Chitas, Rute Malarranha |
| Assunto: | Ciências Naturais::Ciências Biológicas Ciências Médicas::Medicina Clínica |
| Ano: | 2014 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Recombinant protein-based polymers (rPBPs) are emerging as a new class of biomaterials due to their unique chemical, physical and biological properties. With the development of protein engineering and biotechnology, we are now able to use amino acids to design and produce genetically engineered rPBPs with precise control over sequence and length. Indeed, recombinant DNA technology allows the design and biosynthesis of multifunctional complex molecules with exquisite control over structure, making it possible to combine in the same polypeptide chain the properties of two or more different proteins. This opens an unprecedented possibility to create novel compositions for specific applications through the manipulation of the genetic sequence. The silk-elastin-like proteins (SELPs) are rPBPs composed of silk- and elastinlike blocks that combine in the same polypeptide chain the properties of silk and elastin, two remarkable natural proteins. The silk-like unit imparts thermal and chemical stability, whereas the elastin-like unit is a flexible component. This endows SELPs with unique properties that can be explored as base material for the production of advanced functional materials. Leukemia inhibitory factor LIF is a pleiotropic cytokine playing several roles in the regulation of differentiation, cell growth and inflammation, and is an important cytokine for normal keratinocyte growth and wound healing. The functionalization of SELPs can therefore be exploited to create a new generation of functional materials with high potential of application in the biomedical field. The present work was performed in the Department of Biology in University of Minho in the scope of the Master Course in Molecular Genetics. By recurring to recombinant DNA technology techniques, murine recombinant LIF was used to functionalize a SELP copolymer. For this, two constructions were performed considering the fusion of LIF to the N- and C-terminal of the SELP chain. The recombinant functionalized SELPs were successfully expressed in Escherichia coli and purified by a simple and non-chromatographic method with high recovery rates. Furthermore, aiming at its subsequent use in drug delivery systems, production and purification of sole LIF was also considered. The recombinant proteins were assessed for their bioactivity in C2C12 cells showing promising results and suggesting that these recombinant proteins could have a high potential of application in the biomedical field. |
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