Protein expression, purification and assay procedures of hAASS-SDH
Cell line: DH10Bac
Tags and additions: N-terminal, TEV protease cleavable hexahistidine tag
Construct protein sequence:
(underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
Harvested cells were resuspended in lysis buffer (50 mM HEPES pH 7.4, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.4, 0.5 mM TCEP, 1 µL per 1 mL protease inhibitor cocktail EDTA-free).
Cell pellet was dissolved in approximately 200 mL lysis buffer and broken by homogenization by 2 passes at 12,000 psi. The cell debris was pelleted at 35000 x g, 1h and the supernatant used for purification on a gravity flow Ni-NTA column (5 mL).
Buffers used are detailed hereafter;
Binding Buffer: 50 mM HEPES pH 7.4, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.4, 0.5 mM TCEP
Wash Buffer: 50 mM HEPES pH 7.4, 500 mM NaCl, 5% Glycerol, 40 mM Imidazole pH 7.4, 0.5 mM TCEP
Elution Buffer: 50 mM HEPES pH 7.4, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.4, 0.5 mM TCEP
The clarified cell extract was added to 5 ml of Ni-NTA resin pre-equilibrated with lysis buffer and passed through a glass column. The column was then washed with Binding Buffer (2 x 50 mL) and Wash Buffer (2 x 50 mL). The protein was eluted with Elution Buffer in 5 x 5 mL fractions. The eluted fractions from column 1 were pooled and concentrated to 5 mL with a 30 kDa MWCO spin concentrator and injected into an S200 16/60 column (pre-equilibrated in GF Buffer (50 mM HEPES pH 7.4, 500 mM NaCl, 0.5 mM TCEP, 5% Glycerol)) at 1.0 mL/min. 1.5 mL-fractions were collected. The eluted protein was cleaved overnight at 4 °C by TEV protease (1/20 (w/w)). The following day protein sample was loaded onto 0.5ml Ni-sepharose column pre-equilibrated with GF buffer to remove uncleaved protein. Pooled protein fractions were concentrated to 13 mg/mL using a 30 kDa mwco concentrator.
Activity assay and screening
The SDH activity of hAASS was measured by following NAD+ reduction to NADH, taking advantage that the reduced form of NADH is fluorescent when excited with 340 nm light. We adopted the assay into 384-well format, with detection using the PheraStar fluorescence reader (BMG Labtech) (Excitation/Emission = 340/480 nm). This assay gave a linear response with protein concentration up to 150 nM. A typical reaction consists of 100 nM purified enzyme, 0.2 mM NAD+, 1.3 mM saccharopine. The reaction buffer consists of 25 mM HEPES pH 7.4, 100 mM NaCl, 0.1% BSA, 0.05% CHAPS. The compound libraries (LOPAC (Sigma) and NIH Clinical Collections I&II) were screened in-house at 20 μM compound concentration.
Differential scanning fluorimetry
DSF was performed in a 96-well plate using an Mx3005p RT-PCR machine (Stratagene) with excitation and emission filters of 492 and 610 nm, respectively. Each well consisted of 2 µL protein in 2 µM DSF buffer (150 mM NaCl, 10 mM HEPES pH 7.5), 2 µL SYPRO ORANGE diluted 1000-fold in DSF buffer from the manufacturers stock (Invitrogen), and (if applicable) 2 µL ligand at various concentrations. Fluorescence intensities were measured from 25 to 96°C with a ramp rate of 3°C/min.
Apo crystals were prepared by mixing 50 nL of hAASS-SDH protein (80 mg/mL) with 100 nL of reservoir solution containing 20% PEG3350, 0.1M Tris pH 7.5 and 0.2-0.33 M sodium malonate. NAD+-bound crystals were prepared by mixing 100 nL of hAASS-SDH (18 mg/mL, in molar excess of NAD+) with 50 nL of reservoir solution containing 25% PEG3350, 0.2M NaCl and 0.1M tris pH 8.5. Crystals were cryo-protected in 9% butanediol before freezing in liquid nitrogen. For the fragment screening campaign, crystals were soaked with compounds (10/50/500 mM) in the crystallization solution supplemented with 8% butanediol for 5-30 min, and frozen in liquid nitrogen.
Structure determination procedures
hAASS-SDH apo and NAD+-bound crystals belong to different spacegroups (P43212 vs P212121). The structure of hAASS-SDH was solved by molecular replacement with the program PHASER, using the fungal saccharopine reductase from S.cerevisiae (PDB code 2AXQ) as search model (38% sequence identity). Two molecules were found in the asymmetric unit (a.u.). The initial model was rebuilt using phenix.autobuild. A bound NAD+ molecule in the active site was identified by difference Fourier method and manually placed into the electron density using Coot. To complete the model iterative cycles of phenix.refine including TLS refinement followed by manual model building for missing residues using coot were performed. No NCS restraints were applied due to conformational differences in domain III (res 278-376) of the two copies in the a.u. Solvent atoms were placed during the last four rounds of refinement using phenix.refine. For the fragment screening campaign, ligands were identified by DIMPLE (8)(https://github.com/ccp4/dimple) using difference density maps. Weaker binders with low occupancy were evaluated using PANDDA (9)(https://pandda.bitbucket.io/), based on statistical models to find ligand density present in a given dataset that is not present in majority of datasets. Coordinates and structure factors for all data sets are deposited in the RCSB Protein Data Bank. Data collection and refinement statistics are available from the PDB pages.
Commercially available reagents
CRISPR/Cas9 knockout plasmids
SCBT: Cat # sc-406408
Genscript: Cat # 10157