Entry Clone Source: MGC |
Entry Clone Accession: BC014928 |
SGC Construct ID: MINAB-c001 |
GenBank GI number: gi|23346418 |
Vector: pNIC28-Bsa4. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Amplified construct sequence: |
Final protein sequence (tag sequence in lowercase) |
Tags and additions: N-terminal TEV cleavable 6His tag - mhhhhhhssgvdlgtenlyfq*s(m), cleaves at *. Tag removed: Yes |
Host: E. coli BL21(DE3)-R3-pRARE2 |
Growth medium, induction protocol: Medium: TB + 50 µg/ml Kanamycin + 34 µg/ml chloramphenicol. 4 x 1 liter TB in 2.5-L baffled flasks were inoculated with 5 ml overnight culture and grown at 37°C. The protein expression was induced with 0.2 mM IPTG at OD600= 1.2 for 18 h at 18°C. |
Extraction buffer, extraction method: Lysis buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP, PMSF 0.5 mM and 15 units/ml Benzonase. Frozen cell pellets were thawed and resuspended in a total volume of 400 ml lysis buffer. The cells were disrupted by high pressure homogenisation at 15 kpsi (Avestin C5) . Cell debris were removed by centrifugation for 60 minutes at 40,000 x g. |
Column 1: 2 ml Ni-Sepharose 6 FF gravity column |
Column 1 Procedure: The column was equilibrated with 5 column 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 20 mM Imidazole, 0.5 mM TCEP. The lysate was applied to the column and allowed to flow through. The column was washed with 10 column volumes of 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 20 mM Imidazole, 0.5 mM TCEP, then more stringently with 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 40 mM Imidazole, 0.5 mM TCEP. The column was then eluted with 5 column volumes of 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 250 mM Imidazole, 0.5 mM TCEP. TEV protease was added to a final concentration of 60 ug/ml per mg of target protein, the digestion was allowed to proceed overnight at 4°C. |
Column 2: 10/20 HiPrep Desalting column |
Column 2 Procedure: The column was equilibrated with 50 mM HEPES pH 7.5, 50 mM NaCl. The digest was applied to the column and the buffer exchanged protein fraction collected. |
Column 3: 5 ml Q-Sepharose HP |
Column 3 Procedure: The column was equilibrated with 5 column volume 50 mM HEPES pH 7.5, 50 mM NaCl. The sample was applied to the column and the column eluted with a 20 column volume gradient from 50 mM to 500 mM NaCl. 1 ml fractions were collected and analysed by SDS-PAGE. The most pure fractions were pooled. |
Column 4: 10/20 HiPrep Desalting column |
Column 4 Procedure: The column was equilibrated with 10 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol and 0.5 mM TCEP. The sample was applied to the column and the buffer exchanged protein fraction collected. |
Concentration: The purified protein was concentrated to 11.5 mg/ml using an Amicon Ultracel ultrafiltration unit at 4,000 x g. Concentration was determined from absorbance at 280 nm using a NANODROP-1000 spectrophotometer. |
Crystallisation: Crystals were grown at 20°C by vapour diffusion in sitting drops by mixing protein (11.5 mg/ml) and well solution containing 12% PEG 3350; 0.005M CoCl2; 0.005M MgCl2; 0.005M CdCl2; 0.005M NiCl2; 0.1M HEPES pH 7.5 at a protein to precipitant ratio of 2:1. The crystal was cryo-protected using well solution supplemented with 20% (v/v) ethylene glycol and flash-cooled in liquid nitrogen. |
Data collection: Resolution: 2.6 Å; X-ray source: Synchrontron SLS-X10SA. |