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1.
Nat Commun ; 10(1): 2905, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266953

RESUMO

Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (-30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. To discover polyanionic proteins with optimal delivery properties, we evaluate negatively charged natural human proteins for their ability to deliver proteins into cultured mammalian cells and human primary fibroblasts. Here we discover that ProTα, a small, widely expressed, intrinsically disordered human protein, enables up to ~10-fold more efficient cationic lipid-mediated protein delivery compared to (-30)GFP. ProTα enables efficient delivery at low- to mid-nM concentrations of two unrelated genome editing proteins, Cre recombinase and zinc-finger nucleases, under conditions in which (-30)GFP fusion or cationic lipid alone does not result in substantial activity. ProTα may enable mammalian cell protein delivery applications when delivery potency is limiting.


Assuntos
Edição de Genes/métodos , Lipossomos/química , Proteínas/química , Edição de Genes/instrumentação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Integrases/química , Integrases/genética , Integrases/metabolismo , Lipossomos/metabolismo , Transporte Proteico , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismo
2.
Chembiochem ; 19(1): 66-75, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29077265

RESUMO

Application of artificial nucleases (ANs) in genome editing is still hindered by their cytotoxicity related to off-target cleavages. This problem can be targeted by regulation of the nuclease domain. Here, we provide an experimental survey of computationally designed integrated zinc finger nucleases, constructed by linking the inactivated catalytic centre and the allosteric activator sequence of the colicin E7 nuclease domain to the two opposite termini of a zinc finger array. DNA specificity and metal binding were confirmed by electrophoretic mobility shift assays, synchrotron radiation circular dichroism spectroscopy, and nano-electrospray ionisation mass spectrometry. In situ intramolecular activation of the nuclease domain was observed, resulting in specific cleavage of DNA with moderate activity. This study represents a new approach to AN design through integrated nucleases consisting of three (regulator, DNA-binding, and nuclease) units, rather than simple chimera. The optimisation of such ANs could lead to safe gene editing enzymes.


Assuntos
Nucleases de Dedos de Zinco/metabolismo , Domínio Catalítico , Dicroísmo Circular , DNA/química , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Células HEK293 , Humanos , Cinética , Metais/química , Metais/metabolismo , Microscopia de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/genética
3.
Methods Mol Biol ; 1630: 1-24, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28643245

RESUMO

Zinc-finger nucleases (ZFNs) are programmable nucleases that have opened the door to the genome editing era. The construction of ZFNs recognizing a target sequence of interest is laborious, and has not been widely used recently. However, key ZFN patents are expiring over the next 2-4 years, enabling a wide range of deployments for clinical and industrial applications. This article introduces a ZFN construction protocol that uses bacterial one-hybrid (B1H) selection and single-stranded annealing (SSA) assay.


Assuntos
Bactérias/crescimento & desenvolvimento , Técnicas do Sistema de Duplo-Híbrido , Nucleases de Dedos de Zinco/metabolismo , Bactérias/genética , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples , Edição de Genes , Engenharia de Proteínas , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/genética , Dedos de Zinco
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