An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP
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 GFP1-10<sub>fast</sub>. We detail the expression and purification of the GFP1-10<sub>fast</sub> 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.<sup>1</sup>.
Authors
- Anna Maria Giuliodori (ORCID: https://orcid.org/0000-0002-6567-1118)
- Pohl Milón (ORCID: https://orcid.org/0000-0001-6679-5473)
- Attilio Fabbretti (ORCID: https://orcid.org/0000-0003-3928-2781)
- Chiara Poletti (ORCID: https://orcid.org/0009-0003-4402-1137)
- Ana Elena Sanchez-Castro (ORCID: https://orcid.org/0000-0003-3861-3083)
- Therese Manuela Nloh Tientcheu
- Ana Quiroz-Huanca
- Pablo Soriano-Castillo
Institutions
- Università di Camerino (IT)
- Peruvian University of Applied Sciences (PE)
- National University of San Marcos (PE)
Publication Details
- Journal
- STAR Protocols
- Published
- 2024-11-15
- DOI
- https://doi.org/10.1016/j.xpro.2024.103448
- Primary Topic
- RNA Research and Splicing
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- European Commission
- Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica