RESUMEN
Biologics are a promising new class of drugs based on complex macromolecules such as proteins and nucleic acids. However, delivery of these macromolecules into the cytoplasm of target cells remains a significant challenge. Here we present one potential solution: bacterial nanomachines that have evolved over millions of years to efficiently deliver proteins and nucleic acids across cell membranes and between cells. In this review, we provide a brief overview of the different bacterial systems capable of direct delivery into the eukaryotic cytoplasm and the medical applications for which they are being investigated, along with a perspective on the future directions of this exciting field.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Sistemas de Secreción Bacterianos , Productos Biológicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Animales , Portadores de Fármacos , Sistemas de Liberación de Medicamentos/efectos adversos , Sistemas de Liberación de Medicamentos/métodos , Humanos , Inmunoterapia/métodos , Sustancias Macromoleculares/administración & dosificación , Sustancias Macromoleculares/metabolismo , NanopartículasRESUMEN
Proteins are versatile macromolecules with diverse structure, charge, and function. They are ideal building blocks for biomaterials for drug delivery, biosensing, or tissue engineering applications. Simultaneously, the need to develop green alternatives to chemical processes has led to renewed interest in multienzyme biocatalytic routes to fine, specialty, and commodity chemicals. Therefore, a method to reliably assemble protein complexes using protein-protein interactions would facilitate the rapid production of new materials. Here we show a method for modular assembly of protein materials using a supercharged protein as a scaffolding "hub" onto which target proteins bearing oppositely charged domains have been self-assembled. The physical properties of the material can be tuned through blending and heating and disassembly triggered using changes in pH or salt concentration. The system can be extended to the synthesis of living materials. Our modular method can be used to reliably direct the self-assembly of proteins using small charged tag domains that can be easily encoded in a fusion protein.
Asunto(s)
Modelos Moleculares , Proteínas/química , Materiales Biocompatibles/química , Transferencia Resonante de Energía de Fluorescencia , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ingeniería de Proteínas , Mapas de Interacción de Proteínas , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Cloruro de Sodio/química , Electricidad EstáticaRESUMEN
A dual function blue fluorescent protein from Vibrio vulnificus is also an NADPH-dependent oxidoreductase, rendering it a useful tool for biophysical studies.
Asunto(s)
NADH NADPH Oxidorreductasas/metabolismo , Oxidorreductasas/metabolismo , Vibrio vulnificus/enzimología , Vibrio vulnificus/genética , Sitios de Unión , Codón/genética , Escherichia coli/genética , Colorantes Fluorescentes , Cinética , Mutación , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/aislamiento & purificación , Oxidorreductasas/química , Oxidorreductasas/aislamiento & purificación , Tirosina/químicaRESUMEN
Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimization, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterize in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimize pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimization. In summary, EcoFlex provides a standardized and multifunctional kit for a variety of applications in E. coli synthetic biology.