RESUMEN
Nature regulates interference between cellular processes-allowing more complexity of life-by confining specific functions to organelles. Inspired by this concept, we designed an artificial organelle dedicated to protein engineering. We generated a membraneless organelle to translate only one type of messenger RNA-by recruiting an RNA-targeting system, stop codon-suppression machinery, and ribosomes-by means of phase separation and spatial targeting. This enables site-specific protein engineering with a tailored noncanonical function in response to one specific codon in the entire genome only in the protein of choice. Our results demonstrate a simple yet effective approach to the generation of artificial organelles that provides a route toward customized orthogonal translation and protein engineering in semisynthetic eukaryotic cells.
Asunto(s)
Codón/genética , Código Genético , Orgánulos/metabolismo , Orgánulos/ultraestructura , Biosíntesis de Proteínas/genética , Ingeniería de Proteínas/métodos , ARN Mensajero/genética , Animales , Células COS , Caenorhabditis elegans/genética , Membrana Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Lisina/análogos & derivados , Lisina/genética , Methanosarcina , Orgánulos/química , ARN de Transferencia/química , Ribosomas/química , Biología SintéticaRESUMEN
We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure-function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies.
Asunto(s)
Proteínas Fluorescentes Verdes/biosíntesis , Complejos Multiproteicos/biosíntesis , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Virales/biosíntesis , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Técnicas de Cultivo de Célula , Transferencia Resonante de Energía de Fluorescencia/métodos , Código Genético , Vectores Genéticos , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Plásmidos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.
