15n nh4cl

The recombinant plasmids harboring His-tagged or GST-tagged Vav2-SH2 were transformed into Escherichia coli BL21 (DE3) (Novagen) strain. Cells were grown at 37 °C up to an A600 nm of 0.8, and then were induced with 0.5 mM IPTG at 25 °C for 8 h. For the production of uniformly ¹⁵N-labeled samples, cells were grown in minimal medium using ¹⁵NH₄CL (0.5 g/L) as the sole nitrogen source. ¹⁵N-NH₄Cl was purchased from Cambridge Isotope Laboratories, Inc. The His-tagged proteins were purified by a chelated-nickel column followed by Thrombin protease treatment to remove the tag as previous described^{54 (link)}. The GST and GST fusion proteins were purified using immobilized glutathione. All proteins were further purified on a Superdex75 gel-filtration column (GE Healthcare, Piscataway, NJ, USA). The purity of proteins was confirmed by SDS–PAGE. Protein concentrations were estimated with absorbance spectroscopy using the molar absorption coefficient.
The phosphotyrosine peptide APP-pY682 (QNG-pY-ENPT) corresponding to residues 679–686 of APP695 with Y682 phosphorylated and unphosphorylated peptide APP-Y682 (QNGYENPT) were synthesized by GL Biochem Ltd. (Shanghai).

Isotopically enriched chemical compounds, namely [¹⁵N]-NH₄Cl, [¹³C]-glucose and ²H₂O, were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Chromatography columns HiTrap Heparin, HiTrap Q HP and Superdex 75 10/300 GL, the Peristaltic Pump P-1 and AKTA Start were from GE Healthcare (Little Chalfont, UK). Reagents for SDS-PAGE were from BioRad (Hercules, CA, USA). Dialysis membrane tube Spectra/Por of 3.5 kDa nominal cut-off was from Spectrum Laboratories (Los Angeles, CA, USA) and Amicon Ultra-4 or -15 centrifugal filters of 10 or 3 kDa nominal cut-off were from Millipore (Billerica, MA, USA). Unless otherwise described, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Fragments corresponding to the first dsRBD of HYL1 protein (HYL1-1) and their R1, R2 and R3 mutants were amplified by PCR from the binary vectors for plant transformation using the primers shown in Text S1, cloned into the pET-TEV expression vector and sequenced [23] (link). The plasmids were transformed in E. coli BL21 (DE3) cells, which were then grown at 37°C in M9 minimal medium supplemented with 1 g/l ¹⁵N-NH₄Cl (Cambridge Isotope Laboratories) in the case of NMR experiments and in LB medium for the stability and affinity experiments.
Protein expression was induced with 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) at OD₆₀₀ ≈0.7 and cells were grown for 4–5 hours. Cells were harvested, resuspended in a buffer containing 100 mM phosphate, 10 mM Tris, 5 mM β-mercaptoethanol, 8 M Urea pH 8 and disrupted by sonication. The denatured proteins were purified using a Ni (II) column and refolded by dyalisis in 100 volumes of 100 mM phosphate, 50 mM NaCl, 5 mM β-mercaptoethanol, 50 mM glutamate, 50 mM arginine. The refolded proteins were then digested using 1∶100 mass ratio of His-tagged TEV protease, to remove the His-tag, and the protease was removed by a further passage through a Ni (II) column.