Entry Clone Source: MGC |
Entry Clone Accession: IMAGE:3908548 |
SGC Construct ID: MUTA-c602 |
GenBank GI number: gi|4557767 |
Vector: pNIC-CTHF. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Amplified construct sequence: |
Final protein sequence (tag sequence in lowercase) |
Tags and additions: C-terminal hexahistidine tag and Flag tag, TEV cleavable |
Host: BL21(DE3)-R3-pRARE2 |
Expression:10µl of BL21(DE3)-R3 glycerol stock were inoculated into 5ml of Terrific broth medium supplemented with kanamycin (50 µg/ml) and chloramphenicol (34µg/ml) and grown overnight at 37°C, 200rpm. In the morning 1L of TB supplemented with the same antibiotics was inoculated with 10ml of the overnight culture and incubated at 37°C with intensive shaking (160rpm). After the OD600 reached 1.5, the temperature was changed to 18°C and IPTG was added to the final concentration of 1mM. The culture was incubated at 18°C with shaking (160rpm) for additional 18h. The following morning the 14 l culture was harvested and centrifuged for 10min at 4000rpm. Supernatant was discarded and the cell pellets were resuspended in 75ml of a lysis buffer and frozen at -80°C. |
Extraction: Lysis buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5mM Imidazole, 5% glycerol + EDTA-free Complete (1 tablet/50ml). |
Purification: Step 1: Ni-affinity, Ni-Sepharose - (GE Healthcare) Akta-Express; Step 2: Superdex 200 Column, HiPrep 16/60 (Amersham); Step 3: Ion exchange -5ml HiTrap Q Sepharose |
Buffers: Start buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5mM Imidazole, 5% glycerol, 1mM PMSF, 0.5mM TCEP; Washing buffer: 50mM HEPES pH 7.5, 500mM NaCl, 10mM Imidazole, 5% glycerol, 1mM PMSF, 0.5mM TCEP; Elution buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5% glycerol, 250mM Imidazole, 0.5mM TCEP. |
GF buffer: 10mM HEPES pH 7.5, 500mM NaCl, 5% glycerol, 0.5mM TCEP |
IEX buffers: Buffer A: 50mM Tris pH 8.0. Buffer B: 50mM Tris pH 8.0, 1M NaCl. |
Procedure: The cell extract was loaded on the ÄKTA Express system. The extinction at 280nm was monitored and fractions were collected and analyzed by SDS-PAGE. MUTA containing fractions were diluted with IEX buffer A to a concentration of NaCl of 50 mM. The protein was loaded onto a HiTrap Q Sepharose column, and eluted with a NaCl gradient (50-300mM). Fractions containing protein were analysed by SDS-PAGE. |
Concentration and buffer exchange: Using Amicon Ultra-15 concentrators with 30kDa cutoff, the sample was concentrated to 11mg/ml. Concentrations were determined from the absorbance at 280 nm using a NanoDrop spectrophotometer. |
Mass spectrometry characterization: The calculated mass of the construct was 84724 Da, and the observed mass (ESI-MS) was 84510Da, compatible with an N-terminal methionine and serine deletion. |
Crystallisation: Prior to crystallization protein (19.6 mg/ml) was pre-incubated with 250 µM adenosylcobalamin and 2.5 mM malonyl-CoA. Crystals were grown by vapour diffusion at 20°C, in 150 nl sitting drops mixing 100 nl protein and 50 nl mother liquor (20% PEG3350, 0.1 M Bis-Tris pH 5.5, 0.1 M NH4-sulphate) equilibrated against 20 µl reservoir containing mother liquor. Crystals were cryo-protected in mother liquor containing 25% (v/v) ethylene glycol and flash-cooled in liquid nitrogen. |
Data collection: Resolution: 2.10 Å; X-ray source: Swiss Light source (SLS), beamline X-10. |