An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP
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Authors
Quiroz-Huanca, AnaSanchez-Castro, Ana
Soriano-Castillo, Pablo
Poletti, Chiara
Nloh Tientcheu, Therese Manuela
Fabbretti, Attilio
Giuliodori, Anna Maria
Milon, Pohl
Issue Date
2024-12-20Keywords
Biotechnology and bioengineeringChemistry
High-Throughput Screening
Protein expression and purification
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Show full item recordPublisher
Cell PresJournal
STAR ProtocolsDOI
10.1016/j.xpro.2024.103448Abstract
Here, we present an in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of split-GFP for translation test (FAST), based on the small fragment GFP11 binding to GFP1-10fast. We detail the expression and purification of the GFP1-10fast protein, DNA template amplification, in vitro GFP11-tagged CspA synthesis, FAST detection of the GFP11-tagged protein, and optional recovery of the fluorescent complex. In vitro synthesis of GFP11 maximizes the molar yield of synthesized proteins, providing enhanced sensitivity to test translation inhibitors. For complete details on the use and execution of this protocol, please refer to Pham et al.Type
info:eu-repo/semantics/articleRights
info:eu-repo/semantics/embargoedAccessLanguage
engEISSN
26661667Sponsors
Horizon 2020ae974a485f413a2113503eed53cd6c53
10.1016/j.xpro.2024.103448
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