Publicação
Functionalization of spider silk using enzymatic sortase A coupling
| Resumo: | Silk is a natural protein fibre spun by spiders, some insects and silkworms. Spider silk is of interest because of its mechanical strength, elasticity and biocompatibility, in fact, spider silk is considered to be the strongest natural biopolymer known. Recombinant production of spider silk proteins has begun to meet the needs of the growing biotechnological interests, and recombinant production in various hosts has been a widely used approach, with varying results. In search of mimicking the silk proteins, 4RepCT has been engineered with only 4 gly/ala repeats followed by a non-repetitive C-terminal. This protein has been successfully produced in a soluble form using Escherichia coli, yet forming macroscopic silk-like fibres. Studies on 4RepCT fibres have shown biocompatibility and a limited immunological response when subcutaneously implanted in rats. Functionalization is a process where additional peptide motifs, protein domains or organic compounds are coupled to 4RepCT to add new properties for use in medical and biotechnology disciplines. There is, however, currently, a need for new coupling methods, in addition to, for example, manipulation on the genetic level. Therefore, this study was aimed to investigate a new method for functionalization of 4RepCT silk using two model proteins (domain Z and fibroblast growth factor-2) with the help of Sortase A mediated covalent coupling. The results have shown that a Sortase A enzyme with 3 mutations, coupling at ambient temperature and a reaction time between 0.5 and 1.5 h are suitable conditions for efficient coupling of the model proteins to 4RepCT silk coatings. Coupling of the Z domain to silk using the Coupling during coating (CDC) method and FGF2 to silk using the Coupling after coating (CAC) method showed best binding of their target molecules (IgG and FGF2 receptor, respectively). Analysis by Surface Plasmon Resonance was efficient to distinguish these differences in binding rates. |
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| Autores principais: | Pasupuleti, Rajeev |
| Assunto: | Seda Acoplamento enzimático |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Instituto Politécnico de Bragança |
| Idioma: | inglês |
| Origem: | Biblioteca Digital do IPB |
| Resumo: | Silk is a natural protein fibre spun by spiders, some insects and silkworms. Spider silk is of interest because of its mechanical strength, elasticity and biocompatibility, in fact, spider silk is considered to be the strongest natural biopolymer known. Recombinant production of spider silk proteins has begun to meet the needs of the growing biotechnological interests, and recombinant production in various hosts has been a widely used approach, with varying results. In search of mimicking the silk proteins, 4RepCT has been engineered with only 4 gly/ala repeats followed by a non-repetitive C-terminal. This protein has been successfully produced in a soluble form using Escherichia coli, yet forming macroscopic silk-like fibres. Studies on 4RepCT fibres have shown biocompatibility and a limited immunological response when subcutaneously implanted in rats. Functionalization is a process where additional peptide motifs, protein domains or organic compounds are coupled to 4RepCT to add new properties for use in medical and biotechnology disciplines. There is, however, currently, a need for new coupling methods, in addition to, for example, manipulation on the genetic level. Therefore, this study was aimed to investigate a new method for functionalization of 4RepCT silk using two model proteins (domain Z and fibroblast growth factor-2) with the help of Sortase A mediated covalent coupling. The results have shown that a Sortase A enzyme with 3 mutations, coupling at ambient temperature and a reaction time between 0.5 and 1.5 h are suitable conditions for efficient coupling of the model proteins to 4RepCT silk coatings. Coupling of the Z domain to silk using the Coupling during coating (CDC) method and FGF2 to silk using the Coupling after coating (CAC) method showed best binding of their target molecules (IgG and FGF2 receptor, respectively). Analysis by Surface Plasmon Resonance was efficient to distinguish these differences in binding rates. |
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