Hiload 26 60 superdex 75 gel filtration column

The DNA fragments encoding full-length Lpg0393 (GI:52840638) or Lpg0393Δ17 were inserted into the pET22b CPD vector by standard PCR-based cloning methods. This vector was designed to produce these proteins with a C-terminal fusion of the cysteine protease domain (CPD) of the Vibrio cholera MARTX toxin [37 (link)] with a (His)₁₀-tag. Cell lysate was applied onto a gravity flow column containing HisPur Cobalt Resin (Thermo Scientific). The column was washed with Buffer A (20 mM Tris-HCl (pH 7.5), 0.1 M NaCl and 1mM dithiothreitol) and the CPD-(His)₁₀ tag was autolytically cleaved off on this resin by incubation with 100 μM sodium phytate (Sigma Aldrich). The flow-through fraction was further purified using a Hitrap Q anion exchange column (GE Healthcare) and a HiLoad 26/60 Superdex 75 gel filtration column (GE Healthcare) equilibrated with Buffer A. Selenomethionine (SelMet)-labeled Lpg0393Δ17 was obtained by using E. coli B834(DE3) RIL (Novagen), and purified using the same procedure for native Lpg0393Δ17. Final protein sample was concentrated to 20 mg/ml and flash frozen for storage at -80°C.

Perdeuterated H-FABP was recombinantly expressed at the Institut Laue–Langevin (ILL) Deuteration Laboratory in Grenoble, France, and purified based on the procedure described previously (Zanotti et al., 1992 ▸ ). Briefly, Escherichia coli BL21(DE3) strain (Novagen), transformed with the pJexpress411 plasmid containing the synthetic cDNA coding for H-FABP, was over-expressed in perdeuterated minimal medium using d₈-glycerol as carbon source (Artero et al., 2005 ▸ ). A high cell density fed-batch culture was grown at 30°C to an OD₆₀₀ of 8.5. H-FABP expression was then induced by the addition of 0.5 mM IPTG and cells (40 g wet weight) were harvested at an OD₆₀₀ of around 11. H-FABP was purified using 25 ml of Capto Q resin (GE Healthcare). The protein was eluted with 150 mM NaCl, 25 mM Tris-HCl pH 8.0. Finally, H-FABP was purified in a Hiload 26/60 Superdex 75 gel filtration column (GE Healthcare) in 20 mM Tris pH 7.5, 50 mM NaCl. The published crystallization conditions (Young et al., 1994 ▸ ) were optimized in terms of concentration and seeding conditions.

The wt-PKR_1-169 and Δloop-PKR_1-169 complexes were prepared by incubating purified protein in the presence of a 1.1-fold excess of RNA in Buffer 1 for 15 minutes at room temperature. After incubation, the mixture was applied on a HiLoad 26/60 Superdex 75 gel filtration column (2.6 x 60 cm, GE Healthcare Life Sciences, USA). The elution profile for both complexes showed two distinct peaks: a higher molecular weight peak corresponding to RNA-protein complex followed by a lower molecular weight peak corresponding to free RNA. Elution fractions for both complexes were assayed for the presence of RNA-protein complex via in-line spectrophotometer (at 260 and 280 nm simultaneously), and confirmed by native polyacrylamide gel electrophoresis. Fractions containing RNA-protein complex were pooled and concentrated using GE Healthcare Vivaspin 2 concentrators (GE Healthcare Life Sciences, USA). The purity of the complexes was assessed by the native polyacrylamide gel electrophoresis, whereas concentration was determined using the extinction coefficients of protein and RNA. The DLS profiles of wt-PKR_1-169 and Δloop-PKR_1-169 complexes showed no signs of aggregation across the concentration range at which the experiments were performed.