An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP

Ana Quiroz-Huanca; Ana Sanchez-Castro; Pablo Soriano-Castillo; Chiara Poletti; Therese Manuela Nloh Tientcheu; Attilio Fabbretti; Anna Maria Giuliodori; Pohl Milon

doi:10.1016/j.xpro.2024.103448

An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP

Ana Quiroz-Huanca
, Ana Sanchez-Castro
, Pablo Soriano-Castillo
, Chiara Poletti
, Therese Manuela Nloh Tientcheu
, Attilio Fabbretti
, Anna Maria Giuliodori
, Pohl Milon

Research output: Contribution to journal › Article › peer-review

Abstract

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_fast. We detail the expression and purification of the GFP1-10_fast 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.¹

Original language	English
Article number	103448
Journal	STAR Protocols
Volume	5
Issue number	4
DOIs	https://doi.org/10.1016/j.xpro.2024.103448
State	Published - 20 Dec 2024

Keywords

Biotechnology and bioengineering
Chemistry
High-Throughput Screening
Protein expression and purification

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10.1016/j.xpro.2024.103448

Cite this

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title = "An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP",

abstract = "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.1",

keywords = "Biotechnology and bioengineering, Chemistry, High-Throughput Screening, Protein expression and purification",

author = "Ana Quiroz-Huanca and Ana Sanchez-Castro and Pablo Soriano-Castillo and Chiara Poletti and \{Nloh Tientcheu\}, \{Therese Manuela\} and Attilio Fabbretti and Giuliodori, \{Anna Maria\} and Pohl Milon",

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doi = "10.1016/j.xpro.2024.103448",

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T1 - An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP

AU - Quiroz-Huanca, Ana

AU - Sanchez-Castro, Ana

AU - Soriano-Castillo, Pablo

AU - Poletti, Chiara

AU - Nloh Tientcheu, Therese Manuela

AU - Fabbretti, Attilio

AU - Giuliodori, Anna Maria

AU - Milon, Pohl

PY - 2024/12/20

Y1 - 2024/12/20

N2 - 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.1

AB - 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.1

KW - Biotechnology and bioengineering

KW - Chemistry

KW - High-Throughput Screening

KW - Protein expression and purification

UR - https://www.scopus.com/pages/publications/85209135433

U2 - 10.1016/j.xpro.2024.103448

DO - 10.1016/j.xpro.2024.103448

M3 - Artículo

AN - SCOPUS:85209135433

SN - 2666-1667

VL - 5

JO - STAR Protocols

JF - STAR Protocols

IS - 4

M1 - 103448

ER -

An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP

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