Entry Clone Source: MGC |
Entry Clone Accession: IMAGE:4510603 |
SGC Construct ID: MGC45594A-c007 |
GenBank GI number: gi|28557745 |
Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Amplified construct sequence: |
Tags and additions: N-terminal His-tag with TEV protease cleavage site. |
Final protein sequence (tag sequence in lowercase): |
Host: BL21 (DE3)R3-pRARE2 (Phage resistant strain) |
Growth medium, induction protocol:10 µl of a glycerol stock was inoculated into 3ml of LB medium (supplemented with Kanamycin, 50 µg/ul) in a 15 ml culture tube and cultured at 37°C overnight in a shaking incubator (275 rpm). Next day 1 ml of overnight culture was used to inoculate 1 litre of LB medium and grown at 37°C with vigorous shaking (180 rpm) until the culture reaches an OD600 of 1.5. Temperature was reduced to 18°C, and cells were induced with IPTG at a concentration of 1 mM, and cultivated for 16 hrs. Cells were harvested, centrifuged at 6500 rpm for 10 min, and the pellet was stored at -20°C until further use. |
Extraction buffer, extraction method: Thawed cell pellets were dissolved in 30-40 ml of binding buffer (500 mM NaCl, 5%Glycerol, 50 mM HEPES pH 7.5, 5 mM Imidazole). Cells were lysed by sonication (3x 2 minutes) in a 50ml conical tube. After lysis, the cell lysate was centrifuged at 4°C for 45 minutes at 21,000 (rpm). |
Column 1: Ni-NTA resin. |
Buffers: Wash buffer: 500 mM NaCl, 5% Glycerol, 50 mM Tris-HCl pH 7.5, 30 mM Imidazole; Elution buffer: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 250mM Imidazole. |
Procedure: The clear supernatant after centrifugation was passed through a Ni-NTA (2.5ml resin) column twice. The column was washed with 50 ml of wash buffer, and protein was eluted with 15 ml of elution buffer. |
Column 2: Hiload 16/60 Superdex 200 prep grade 120 ml, GE Healthcare. |
Buffers: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP. |
Procedure: The eluted fractions from the Ni-affinity HisTrap columns were loaded on the gel filtration column in GF buffer at 1.0 ml/min. Eluted proteins were collected in 1 ml fractions. |
Mass spectrometry characterization: corresponds to theoretical mass, as determined by ESI-TOF MS. |
Protein concentration: Protein was concentrated to 5 mg/ml using Vivaspin 10K concentrators. |
Crystallization: Crystals were grown by vapour diffusion in sitting drop at 20°C. Before crystallization setup protein was incubated with 5mM of NADP and 2.5mM of Diclofenac. A sitting drop consisting of 50 nl protein and 50 nl well solution was equilibrated against well solution containing 25% 1,2 propandiol; 10% glycerol; 0.1M Na/K-PO4 pH 6.2. Crystals were mounted in the presence of 25% glycerol and flash-cooled in liquid nitrogen. |
Data Collection: Resolution: 1.9 Å; X-ray source: FRE superbright, single wavelength. |