| Materials and Method | |
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Entry Clone Source: Site-directed mutagenesis |
Entry Clone Accession: n/a |
SGC Construct ID: PHF8A-c380 |
GenBank GI number: gi|32698700 |
Vector: pNH-TrxT |
Coding DNA sequence: |
Tags and additions: N-terminal His6/Thioredoxin -tags with TEV protease cleavage site |
Protein sequence (Tag sequence in lowercase): |
mhhhhhhssgmsdkiihltddsfdtdvlk |
Host: BL21(DE3)-R3-pRARE2 |
Expression protocol: Glycerol stock was used to inoculate 120 ml of LB medium (Luria Broth) supplemented with 50 µg/ml kanamycin and 35 µg/ml chloramphenicol. This starter culture was grown overnight at 37°C and used to inoculate 12 x 1 liter LB culture supplemented with 50 µg/ml kanamycin only. The cells were cultured at 37°C with vigorous shaking (160 rpm) until an OD600 of 0.5-0.6. At that point cells were induced with 0.1 mM IPTG and cultured o/n at 18°C. Cells were harvested at 9000 x g for 10 minutes and the cell pellet of 12L was resuspended in 240 ml of lysis buffer and stored at -80°C until further use. Lysis buffer: 500 mM NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 20 mM imidazole, Complete EDTA-free protease inhibitor (Roche, 1tablet / 50ml). |
Extraction method: Cell pellets from 12 liter previously re-suspended, were thawed and lysed by sonication, followed by centrifugation at 4°C for 45 minutes at 48 000 x g. |
The supernatant was further clarified by filtration (0.45 mm) before loaded on the 5 ml HisTrap FF, column (GE heathcare). |
Column 1: Ni-affinity, HisTrap FF, 5 ml (GE heathcare) |
Buffers: |
Procedure: The centrifuged cell extract was loaded on the column at 5 ml/min on an AKTA-Xpress system (GE heathcare). The column was then washed with 10 column volumes of lysis buffer, 10 column volumes of wash buffer, and then eluted with 3 column volume of elution buffer at 5 ml/min. . The eluted protein was collected and analyzed by SDS-PAGE. |
TEV cleavage: To the fraction containing PHF8A (in total 48.5 mg) 240 µl of TEV protease (6 mg/ml) was added. Cleavage was performed overnight at 4°C and examined by SDS-PAGE. |
Column 2: Ni-NTA |
Procedure: TEV-cleaved protein was exchanged into GF buffer, before being passed through 250 µl of Ni-NTA resin, to remove the TEV protease. The flow through was concentrated to 5 ml using Amicon Ultra 30k, Millipore concentrator and loaded on the Hiload 16/60 Superdex 200 column pre-equilibrated with GF buffer. |
Column 3: Size Exclusion Chromatography (SEC) Hiload 16/60 Superdex 200 prep grade 120 ml (GE/Amersham Biosciences) |
Procedure: Eluted proteins were collected in 1.8 ml fractions and analyzed on SDS-PAGE. |
Buffers: |
Column 4: Ion exchange chromatography, HiTrap 5 ml QP |
Procedure: Fraction containing PHF8A were pooled, diluted to 50 mM NaCl and loaded on the HiTrap 5ml QP pre-equilibrated with buffer A. |
Buffers: |
Concentration: To the fractions containing PHF8A 5% glycerol was added and the protein was concentrated using centricon with 30 kDa cut off (Amicon Ultra 30k, Millipore) to 12.1 mg/ml. |
Mass spectrometry characterization: LC-ESI-MS TOF confirmed the expected mass of 42.909 Da. |
Crystallization: Prior to crystallization, protein was pre-incubated with 2mM N-oxalylglycine at 4oC. Crystals were grown at 4oC by vapour diffusion in sitting drops by mixing protein (12 mg/ml + 2 mM N-oxalylglycine) and well solution containing 1.5 M (NH4)2SO4 and 0.1 M sodium acetate pH 4.25 in a 2:1 ratio. Crystals were cryo-protected using 30% (v/v) glucose and flash-cooled in liquid nitrogen. |