RESUMO
The aim of this study was to develop a transcription activator-like effector (TALE)-based technology to regulate protein synthesis in cell-free systems. We attempted to regulate the T7 promoter system, which has no natural mechanism of expression control, and sought to arbitrarily induce protein expression through the formation and dissociation of TALE and target DNA complexes. Protein synthesis was performed in a cell-free system in the presence of TALE, which recognized and bound to a sequence upstream of the T7 promoter, and protein expression was suppressed by approximately 80 % compared to in the absence of TALE. This suggests that masking part of the promoter region strongly suppresses protein synthesis. Additionally, competitive inhibition of TALE binding to the target DNA template led to protein synthesis levels that were equivalent to the levels in the absence of TALE. Our results demonstrate that DNA recognition by TALE can regulate the expression of the T7 promoter system.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Proteínas Repressoras/metabolismo , Fagos T/fisiologia , Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Proteínas Virais/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Regulação Viral da Expressão Gênica , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/genética , Efetores Semelhantes a Ativadores de Transcrição/genética , Transcrição Gênica , Proteínas Virais/genéticaRESUMO
The conjugation of biomolecules, such as protein, sugar, and DNA, with metal nanoparticles is an important technique for bioassay and biomaterial preparation. In this study, we aim to enzymatically immobilize a functional peptide on gold nanoparticles (AuNPs) using a single-step reaction. We used tyrosinase, a catechol oxidase, to immobilize an enzymatic peptide. We performed immobilization experiments of a fluorescent compound-linked caspase-3 substrate peptide using tyrosinase on chitosan-coated AuNPs. Peptides were effectively immobilized onto the AuNPs depending on the presence of tyrosine within the sequence, which suggests the DOPA-quinone produced from tyrosine, via tyrosinase, is connected to the chitosan amino group. Although fluorescent emission from the immobilized capase-3 substrate was quenched by AuNPs, fluorescence intensity recovery occurred due to the addition of caspase-3. Thus, we were able to easily prepare functional AuNPs that can be used for a caspase-3 activity assay. Our results indicate that the tyrosinase-mediated peptide link to chitosan-coated particles is a useful technique for preparing functionalized nanoparticles.
