TSTA3A (4B8W) Materials & Methods |
Entry clone source: MGC |
SGC Construct ID: TSTA3A-c013 |
Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]. |
DNA sequence: |
Final protein sequence (His6 affinity Tag sequence in lowercase): ^ TEV protease recognition site |
Tags and additions: Cleavable N-terminal His6 tag |
Host: BL21(DE3)-R3-pRARE2. Phage-resistant strain. |
Transformation: The construct DNA was transformed into competent cells of the expression strain by a standard heat shock procedure. |
Glycerol stock preparation: One colony from the transformation was used to inoculate 1 ml of LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day glycerol stocks were prepared from this overnight culture. |
Expression: 5 µl glycerol stock was used to inoculate 50 ml of TB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day this starter culture was used to inoculate 6L of TB media (1 ml starter culture used per 1L) containing 50 µg/ml kanamycin. When the OD600 reached approximately 1.0 the temperature was reduced to 18°C and the cells were induced by the addition of 0.2 mM IPTG. The expression was continued overnight at 18°C. |
Cell harvest: Cells were harvested by centrifugation at 5000 rpm for 11 min at 4°C after which the supernatant was discarded and the cell pellet was frozen at -20°C until future use. |
Cell Lysis: Cell pellets from 6 liter expression were slowly thawed on ice. Afterwards the cell pellets were dissolved in approximately 30-40 ml binding buffer and broken by using an Avestin C-5 homogenizer. The samples were passed through the homogenizer at least three times or until the lysate lost viscosity. After lysis the pellet was separated from the supernatant by centrifugation at 4°C for 45 min at 16,500 rpm. The clear supernatant was transferred to a fresh 50 ml Falcon tube for further purification. |
Column 1: Ni-NTA (2.5 ml volume in a gravity-flow column). |
Column 1 Buffers: |
Column 1 Procedure: The clarified cell extract was further purified on a 2.5 ml of Ni-NTA column. The supernatant, pre-equilibrated with binding buffer, was applied on the column twice before washing and eluting. During the wash step ten times 5 ml portions of wash buffer were added to the column. The protein was eluted with three times 5 ml of Elution Buffer |
Column 2: Superdex 200 10/300 column |
Column 2 Buffers: |
Column 2 Procedure: The eluted fractions from column 1 were pooled separately and concentrated to 5 ml with a 30 kDa mwco spin concentrator. The 5 ml protein sample was injected onto the Superdex 200 column pre-equilibrated with gel filtration buffer, and 1 ml fractions were collected at 1.0 ml/min. The protein eluted between 85 ml and 100 ml column volume. |
Concentration: The eluted protein was concentrated and 50 µl aliquots at a concentration of 12 mg/ml were stored at -80°C. |
Mass spec characterization: |
Crystallization: Crystals were grown by vapour diffusion in hanging drop at 20°C by setting up 12 mg/ml of protein in the presence of 5 mM NADP+ and 10 mM GDP. Pyramidal crystals appeared in a hanging drop consisting of 1 µl protein and 1 µl well solution which had been equilibrated against 500 µl well solution containing 25% PEG 3350, 0.1 M bis-tris-propane pH 7.5, 0.3 M NaBr and 10% (v/v) ethylene glycol. Crystals were mounted in the presence of 30% ethylene glycol and flash cooled in liquid nitrogen. |
Data Collection: Resolution: 2.70 Å |