Entry clone source: Purely Proteins |
Entry clone accession: gi|90652860 |
Final protein sequence: |
Vector: pGEX-6P2. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Tags and additions: Tag sequence: |
Host: BL21 (DE3) phage resistant |
Growth medium, induction protocol: Starter cultures from freshly transformed colonies in 10 ml LB, and ampicillin were grown overnight. This was diluted 1:1000 in fresh media (6L) and was grown at 37°C to an OD 600 of 0.3 and than transferred to 18°C. Expression was induced at an OD 600 of 0.8 using 1 mM IPTG. Cells were harvested after 3h by centrifugation, transferred to 50-ml tubes, and frozen in liquid nitrogen. |
Extraction buffer, extraction method: Extraction buffer: 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 10 mM DTT. The cell pellets (20 g wet wt) were re-suspended in 50 ml extraction buffer, and lysed by a high pressure cell disrupter. Supernatant was centrifuged for 30 minutes at 60,000 rpm. |
Column 1: Glutathione Sepharose 4B affinity, 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer. |
Buffers: Binding buffer: 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 10 mM DTT. |
Procedure: Supernatant was applied at gravity flow, followed by a wash with 30 ml binding buffer. The GST-fusion was cleaved while bound to the column by addition of PreScission protease. The column was gently rotated overnight a 4°C then protein eluated with 3 bed volumes of binding buffer. |
Column 2: SEC |
Buffers: 10 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM DTT. |
Procedure: Akta-Prime |
Column 3: HiTrap Q |
Protein concentration: Centricons 10 kDa cut off |
Mass spec characterisation : LC-ESI-MS TOF confirmed the correct mass expected for this construct (33252 Da). |
Crystallization: Crystals were obtained using sitting drop method at 4°C. Drops were prepared using 150 nl of protein (10 mg/ml concentration) and 150 nl of the well solution (25% PEG 3350, 0.2M LiSO 4 , 100 mM Bis Tris Propane pH 5.5). |
Data Collection: Resolution: 1.8 Å; X-ray source: Synchrontron BESSY BL1. |