Tnt t7 coupled wheat germ extract system

Recombinant SE and GFP proteins fused to maltose-binding protein (MBP) were purified from Escherichia coli as described before (11 ). [³⁵S]-methionine labeled HEN2, ZCCHC8A, ZCCHC8B and RBM7 proteins were obtained by in vitro transcription and translation using the TNT T7 coupled wheat germ extract system (Promega) following the protocol supplied by the manufacturer. Purified MBP-SE and MBP-GFP were bound to amylose beads (NEB) by overnight incubation at 4°C. Excess protein was removed by washing the beads three times with 20 mM Tris HCl pH 7.5, 200 mM NaCl, 1 mM EDTA, cOmplete™ EDTA-free protease inhibitor (Roche) before the radiolabeled proteins were added. After incubation for 2 h at 4°C the beads were washed five times and proteins were eluted with 20 mM maltose. Eluted proteins were separated by 12% SDS-PAGE and detected using a FLA-5000 phosphoimager (Fujifilm).

Full-length PRN1 and GPA1 templates were prepared, amplified and purified as described previously [21] (link). PRN1 and GPA1 protein were separately produced in coupled in vitro transcription/translation reactions using the TNT T7 Coupled Wheat Germ Extract System (Promega; Madison, WI) as directed, with a 90 min translation time at 30°C, and as previously described, with 50% concentration of extract by using a microcentrifugation concentrating filter to maximize retention of protein 30 kD and above (Millipore, Billerica, MA) [29] (link).

To determine the in vitro toxicity of DON, NIV, and NIV3G, commercial in vitro transcription/translation systems [TnT^® T7 Coupled Wheat Germ Extract System and TnT^® T7 Coupled Reticulocyte Lysate System (Promega, Madison, WI, USA)] were used. Transcription/translation reactions were performed as described in Varga et al. (2015) (link) with one minor change being that reactions with rabbit reticulocyte lysate were stopped after 20 min (instead of 24 min) reaction time. At least three independent assays using individual dilutions were performed for each substance.

Total RNA was isolated from fetal liver cells using Qiagen RNeasy Plus Micro kit (74034, Qiagen) according to manufacturer's protocol with following modifications: fetal liver cells were resuspended in 600 μl RLT buffer by vortexing and lysed by passing them through a 23G needle for several times. Total RNA was eluted in 30 μl RNase‐free water. cDNA was prepared using High‐Capacity RNA‐to‐cDNA Kit (4387406, Applied Bioscience) according to the manufacturer's protocol using 1 μg total RNA. PCR on cDNA was performed using Phusion High‐Fidelity DNA polymerase (M0530, NEB) to amplify exon constructs and cloned into pCS2‐T7‐mCherry (kind gift from T.U. Mayer, University of Konstanz, Germany) to generate IVT. Non‐radioactive IVT was performed using the TNT T7‐coupled wheat germ extract system (L4140, Promega), according to the manufacturer’s protocol, except that the reaction time was increased to 3 h. Five microliter IVT was used for immunoblotting as described below.

In vitro toxicity of DON and sulfate derivatives was examined using a commercial in vitro transcription/translation system (TnT^® T7 Coupled Wheat Germ Extract System; Promega, Madison, WI, USA). Standard transcription/translation reactions were performed in a total volume of 15 μL according to the manufacturer’s instructions in the presence of the respective compounds in 0.4 % methanol (final concentration). Ribosomes were first preincubated at 30 °C with inhibitors, buffer, amino acids, and DNA. After 7 min, T7-RNA polymerase was added to start the coupled in vitro transcription/translation reactions, which were stopped after 30 min by adding 1 μL of a 1 mM cycloheximide solution. Efficiency of translation was determined by measuring the activity of the firefly luciferase reporter using the Promega Steady-Glo^® Luciferase Assay System and the EnSpire^® 2300 Multimode Plate Reader from PerkinElmer. Three independent assays using individual dilutions were performed for each substance.