RESUMO
The wide variety of enzymatic pathways that can benefit from enzyme scaffolding is astronomical. While enzyme co-localization based on protein, DNA, and RNA scaffolds has been reported, we still lack scaffolds that offer well-defined and uniform three-dimensional structures for enzyme organization. Here we reported a new approach for protein co-localization using naturally occurring protein nanocages as a scaffold. Two different nanocages, the 25 nm E2 and the 34 nm heptatitis B virus, were used to demonstrate the successfully co-localization of the endoglucanase CelA and cellulose binding domain using the robust SpyTag/SpyCatcher bioconjugation chemistry. Because of the simplicity of the assembly, this strategy is useful not only for in vivo enzyme cascading but also the potential for in vivo applications as well.
Assuntos
Biotecnologia/métodos , Enzimas , Nanoestruturas/química , Proteínas , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Celulossomas/química , Celulossomas/metabolismo , Enzimas/química , Enzimas/metabolismo , Nanotecnologia , Proteínas/química , Proteínas/metabolismoRESUMO
We report a new modular strategy to assemble dCas9-guided enzyme cascades by employing orthogonal post-translation chemistry. Two orthogonal SpyCatcher and SnoopCatcher pairs were used for the one-pot enzyme bioconjugation onto two different dCas9 proteins to enable their guided assembly onto a DNA scaffold. The resulting two-component cellulosomes exhibited 2.8-fold higher reducing sugar production over unassembled enzymes. This platform retains the high binding affinity afforded by dCas9 proteins for easy control over enzyme assembly while offering the flexibility for both in vivo and in vitro assembly of a wide array of enzyme cascades with minimal optimization.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/química , Celulossomas/química , Celulossomas/metabolismo , DNA/química , DNA/metabolismoRESUMO
Nature has evolved a wide range of strategies to create self-assembled protein nanostructures with structurally defined architectures that serve a myriad of highly specialized biological functions. With the advent of biological tools for site-specific protein modifications and de novo protein design, a wide range of customized protein nanocarriers have been created using both natural and synthetic biological building blocks to mimic these native designs for targeted biomedical applications. In this review, different design frameworks and synthetic decoration strategies for achieving these functional protein nanostructures are summarized. Key attributes of these designer protein nanostructures, their unique functions, and their impact on biosensing and therapeutic applications are discussed.
Assuntos
Portadores de Fármacos/química , Nanoestruturas/química , Proteínas/química , Técnicas Biossensoriais/métodos , Ferritinas/química , Ácidos Nucleicos/química , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/químicaRESUMO
Here we reported a new strategy to construct synthetic metabolons using dCas9-guided assembly. Three orthogonal dCas9 proteins were exploited to guide the independent and site-specific assembly of their fusion partners onto a single DNA scaffold. This new platform was applied towards the construction of a two-component cellulosome. Because of the superior binding affinity, the resulting structures exhibited both improved assembly and reducing sugar production. Conditional enzyme assembly was made possible by utilizing toehold-gated sgRNA (thgRNA), which blocks cellulosome formation until the spacer region is unblocked by a RNA trigger. This platform is highly modular owing to the ease of target synthesis, combinations of possible Cas9-fusion arrangements, and expansion to other metabolic pathways.
