Publicação
Development of a molecular biology platform for the production of antimicrobial peptides
| Resumo: | Antimicrobial peptides (AMPs) are important antibiotic candidates due to their reduced or even absent propensity to induce development of resistance from microbes. This fact is important due to the rise of multi-resistant bacteria. This work aims at developing a platform for the production and purification of AMPs using elastin-like polypeptides (ELPs) for the non-chromatographic purification and inteins as self-cleaving tags for the isolation of the AMPs. As general molecular cloning strategy, different adapters (total of nine) were designed and used for the genetic construction of four different expression vectors. Two vectors, named pA1C and pA3N, were designed to insert both ELP (A60) and different AMPs (ABP-CM4; HAMP; BMAP-28) on different sides of the intein. The vector pA1C uses a mutant intein ΔI-CM with the N-terminal cleavage blocked by a point mutation. The pA3N vector, on the other hand, uses a mutant intein ΔI-SM with the C-terminal cleavage blocked by a point mutation. Both inteins are mutants of the Mtu recA intein. As proof of concept for the molecular design, the ABP-CM4 peptide was inserted at the C-terminal in the pA1C vector and at the N-terminal in the pA3N vector. Following genetic constructions, the fusion proteins were expressed in E. coli BL21(DE3) and purified by inverse temperature cycling. After purification, the N-terminal cleaving of the ΔI-SM (mutated) intein was triggered by addition of the reducing agent DTT. The C-terminal cleaving of the ΔI-CM (mutated) intein was triggered with a cleaving solution at pH 6. Although both constructions demonstrated to be able to self-cleave the AMP, the pA3N vector showed to be more promising than pA1C vector, with the latter displaying residual splicing. Additional constructions, with and without His-tags, involved the use of the split intein Npu DnaE aiming at the concatenation/multimerization of biopolymers such as ELPs and silk-elastin-like proteins (SELPs). The genetic constructions explore the use of FRB and FKBP dimerizing domains fused to split inteins to trigger splicing and concatenation after addition of rapamycin. The expression vectors were termed as pA2H (with His-tag) and pA2∅ (without His-tag). Results with A60 demonstrated the concatenation of the biopolymer leading to the formation of molecules with higher molecular weight, although at an uncontrolled rate. |
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| Autores principais: | Alves, João Fernando Machado |
| Assunto: | Concatenation Intein Antimicrobial peptide Splicing Concatenação Inteína Péptido antimicrobiano Ciências Naturais::Ciências Biológicas |
| Ano: | 2019 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Antimicrobial peptides (AMPs) are important antibiotic candidates due to their reduced or even absent propensity to induce development of resistance from microbes. This fact is important due to the rise of multi-resistant bacteria. This work aims at developing a platform for the production and purification of AMPs using elastin-like polypeptides (ELPs) for the non-chromatographic purification and inteins as self-cleaving tags for the isolation of the AMPs. As general molecular cloning strategy, different adapters (total of nine) were designed and used for the genetic construction of four different expression vectors. Two vectors, named pA1C and pA3N, were designed to insert both ELP (A60) and different AMPs (ABP-CM4; HAMP; BMAP-28) on different sides of the intein. The vector pA1C uses a mutant intein ΔI-CM with the N-terminal cleavage blocked by a point mutation. The pA3N vector, on the other hand, uses a mutant intein ΔI-SM with the C-terminal cleavage blocked by a point mutation. Both inteins are mutants of the Mtu recA intein. As proof of concept for the molecular design, the ABP-CM4 peptide was inserted at the C-terminal in the pA1C vector and at the N-terminal in the pA3N vector. Following genetic constructions, the fusion proteins were expressed in E. coli BL21(DE3) and purified by inverse temperature cycling. After purification, the N-terminal cleaving of the ΔI-SM (mutated) intein was triggered by addition of the reducing agent DTT. The C-terminal cleaving of the ΔI-CM (mutated) intein was triggered with a cleaving solution at pH 6. Although both constructions demonstrated to be able to self-cleave the AMP, the pA3N vector showed to be more promising than pA1C vector, with the latter displaying residual splicing. Additional constructions, with and without His-tags, involved the use of the split intein Npu DnaE aiming at the concatenation/multimerization of biopolymers such as ELPs and silk-elastin-like proteins (SELPs). The genetic constructions explore the use of FRB and FKBP dimerizing domains fused to split inteins to trigger splicing and concatenation after addition of rapamycin. The expression vectors were termed as pA2H (with His-tag) and pA2∅ (without His-tag). Results with A60 demonstrated the concatenation of the biopolymer leading to the formation of molecules with higher molecular weight, although at an uncontrolled rate. |
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