ArticleProtein science : a publication of the Protein Society2010
TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector.
Article in Protein science : a publication of the Protein Society, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed.
- Efficient production of human mitochondrial chaperonin (Hsp60/Hsp10) in Escherichia coli using a polyprotein strategy.Protein expression and purification · 2025Article
- Efficient production of protein complexes in mammalian cells using a poxvirus vector.PloS one · 2022Article
- Expression of in vivo biotinylated recombinant antigens SAG1 and SAG2A from Toxoplasma gondii for improved seroepidemiological bead-based multiplex assays.BMC biotechnology · 2020Article
- Tobacco etch virus (TEV) protease with multiple mutations to improve solubility and reduce self-cleavage exhibits enhanced enzymatic activity.FEBS open bio · 2020Article
- Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology.Proceedings of the National Academy of Sciences of the United States of America · 2018Article
- Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX.Journal of visualized experiments : JoVE · 2017Article
- Post-translational control of genetic circuits using Potyvirus proteases.Nucleic acids research · 2016Article
- Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.Nanomedicine : nanotechnology, biology, and medicine · 2016Article
- Article
- Insight into the structural stability of wild type and mutants of the tobacco etch virus protease with molecular dynamics simulations.Journal of molecular modeling · 2013Article
- An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease.Protein & cell · 2012Article
- MultiBac: expanding the research toolbox for multiprotein complexes.Trends in biochemical sciences · 2012Review
- Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells.Journal of structural biology · 2011Review
- SmartBac, a new baculovirus system for large protein complex production.Journal of structural biology: XArticle
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3 authors.
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Abstract
Delivery and expression of multiple genes is an important requirement in a range of applications such as the engineering of synthetic signaling pathways and the induction of pluripotent stem cells. However, conventional approaches are often inefficient, nonstoichiometric and may limit the maximum number of genes that can be simultaneously expressed. We here describe a versatile approach for multiple gene delivery using a single expression vector by mimicking the protein expression strategy of RNA viruses. This was accomplished by first expressing the genes together with TEV protease as a single fusion protein, then proteolytically self-cleaving the fusion protein into functional components. To demonstrate this method in E. coli cells, we analyzed the translation products using SDS-PAGE and showed that the fusion protein was efficiently cleaved into its components, which can then be purified individually or as a binding complex. To demonstrate this method in mammalian cells, we designed a differential localization scheme and used live cell imaging to observe the distinctive subcellular targeting of the processed products. We also showed that the stoichiometry of the processed products was consistent and corresponded with the frequency of appearance of their genes on the expression vector. In summary, the efficient expression and separation of up to three genes was achieved in both E. coli and mammalian cells using a single TEV protease self-processing vector.
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