TY - JOUR
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
N1 - Publisher Copyright:
© 2024 The Author(s)
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 -