Entry Clone Source: MGC |
Entry Clone Accession: IMAGE:5087393 |
SGC Construct ID: HPDA-c103 |
GenBank GI number: gi|4504477 |
Vector: Vector: pNIC-CTHF. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Amplified construct sequence: |
Final protein sequence (tag sequence in lowercase): |
Tags and additions: C-terminal, TEV cleavable hexahistidine tag. |
| Host: E. coli BL21(DE3)-R3-pRARE2 |
Expression: 10ul of BL21(DE3)-R3-pRARE2 glycerol stock were inoculated into 5ml of TB with 50ug/ml of kanamycin and 34ug/ml chloramphenicol and grown overnight at 37°C, 200rpm. 10ml of overnight culture were added to 1L of TB with 50ug/ml kanamycin and incubated at 37°C, 160rpm. After the OD600 reached 1.0, the temperature was dropped to 18°C and 500ul of 1M IPTG was added to the final concentration of ~0.5mM. The culture was then incubated with shaking overnight at 18°C, 160rpm. The following morning the 4L culture was harvested and centrifuged for 10min at 4000rpm. Supernatant was discarded and cell pellets were resuspended in 80ml of a lysis buffer and frozen at -80°C. |
Extraction: Lysis buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5mM Imidazole, 5% glycerol + 1mM PMSF. The thawed cells were broken by 5 passes at 16.000 psi through a high pressure homogeniser followed by centrifugation for 45 min at 15.000rpm. |
Column 1: Ni-affinity, His-Trap, 1 ml (Amersham) |
| Buffers: Binding buffer: 50mM HEPES pH 7.5, 500mM NaCl, 20mM Imidazole, 5% glycerol, 1mM PMSF, 0.5mM TCEP; Washing buffer: 50mM HEPES pH 7.5, 500mM NaCl, 40mM 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 |
| Procedure: The cell extract was loaded on the AKTA Express system The extinction at 280nm was monitored and fractions were collected and analyzed by SDS-PAGE. Positive fractions were pooled for TEV cleavage. TEV cleavage: The His-tag was cleaved with 1 mg TEV per 40 mg target protein at 4°C overnight. The protein was further purified on IMAC Sepharose using buffers as above. |
Concentration and buffer exchange: |
| Mass spectrometry characterization: Calculated mass of the construct was 45105. The exact mass for the protein lacking the N-terminal methionine was confirmed by the mass spectrometry. |
| Crystallization: Crystals were grown by vapour diffusion in sitting drops at 4°C. A sitting drop consisting of 50 nl protein and 100 nl well solution was equilibrated against well solution containing 0.2M Na(acetate); 0.1M Bis-Tris Propane pH 6.5; 20% PEG 3350, 10% Ethylene Glycol. Crystals were cryo-protected in 10% Ethylene Glycol and 90% well solution before flash-cooling in liquid nitrogen. |
| Data Collection: Resolution: 1.75Å; X-ray source: Diamond microfocus |