Work

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

Year: 2024

Type: article

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 • MBP:GFP1-10 fast protein expression and purification by MBP affinity chromatography (AC) • Steps for obtaining a DNA template using CspA:GFP11 amplicon • Cell-free protein synthesis of CspA:GFP11 and fluorescent detection of split-GFP • Monitoring of mRNA translation inhibition and recovery of protein complexes by AC Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2024.103448