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Entry Clone Source: MGC

Entry Clone Accession: IMAGE:6064429

SGC Construct ID: PTDSRB-c200

GenBank GI number: gi|45219814

Vector: pET-28a(+)

Final protein sequence:
mgsshhhhhhssglvprgshMNHKSKKRI
REAKRSARPELKDSLDWTRHNYYESFSLS
PAAVADNVERADALQLSVEEFVERYERPY
KPVVLLNAQEGWSAQEKWTLERLKRKYRN
QKFKCGEDNDGYSVKMKMKYYIEYMESTR
DDSPLYIFDSSYGEHPKRRKLLEDYKVPK
FFTDDLFQYAGEKRRPPYRWFVMGPPRSG
TGIHIDPLGTSAWNALVQGHKRWCLFPTS
TPRELIKVTRDEGGNQQDEAITWFNVIYP
RTQLPTWPPEFKPLEILQKPGETVFVPGG
WWHVVLNLDTTIAITQNFASSTNFPVVWH
KTVRGRPKLSRKWYRILKQEHPELAVLAD
SVDLQESTGIASDSS

Tag sequence: N-terminal His6 tag, followed by a thrombin protease cleavage site: mgsshhhhhhssglvpr*gsh (* - thrombin cleavage site)

Tag removed: no

Host: BL21(DE3)-R3-pRARE2 (previously known as Rosetta)

Expression protocol: A glycerol stock of E.coli host strain Rosetta carrying the expression plasmid was used to inoculate 10 ml of media supplemented with 25 µg/ml kanamycin and 30 µg/ml chloramphenicol. This starter culture was grown overnight at 37°C and used to inoculate 4L Le Master media prepared with 50 mg/l L-selenomethionine instead of L-met and supplemented with 25 μg/ml kanamycin and 30 μg/ml chloramphenicol. Cells were grown at 37 °C until they reached an OD₆₀₀ of 0.6; the temperature was then reduced to 25 °C and IPTG was added at a final concentration of 0.5 mM. Cells were harvested 16-18 hours post-induction and stored at -80°C.

Lysis buffer: 50 mM Tris, pH 7.5, 200 mM NaCl, 10 mM imidazole, 10% glycerol, 0.2 mM TCEP.

Procedure: Cell pellets were resuspended in 150 ml lysis buffer supplemented with DnasI and lysed by sonication using a Sonics Vibracell sonicator and five 30 s bursts at 60%. Cell debris was removed by centrifugation at 20,000 rpm for 30 min. at 4°C.

Column 1: HisTrap HP 5ml (GE Healthcare)

Buffers:
Lysis buffer: 50 mM Tris, pH 7.5, 200 mM NaCl, 10 mM imidazole, 10% glycerol, 0.2 mM TCEP
Ni-NTA Elution buffer: 50 mM Tris, pH 7.5, 200 mM NaCl, 10% glycerol, 0.2 mM TCEP, 500 mM imidazole
Storage Buffer: 50 mM Tris, pH 7.5, 200 mM NaCl, 10% glycerol, 0.2 mM TCEP.

Procedure: The clarified supernatant was loaded onto a HisTrap column at 2 ml/min using an Akta purifier. The column was developed using a gradient from 0 to 100% elution buffer.  Fractions were analysed by SDS-PAGE and those containing protein of the correct molecular weight and the best purity were pooled and concentrated using a 10,000 MWCO filter (Millipore).

Protein concentration: The protein solution was buffer-exchanged into storage buffer using a 10,000 MWCO filter and concentrated to 19 mg/ml measured by a NanoDrop ND-1000 spectrophotometer. Protein was aliquoted and stored at -80°C.

Crystallization: Crystals were grown at 4°C by vapour diffusion in sitting drops by mixing protein (19 mg/ml) and well solution containing 0.8M NaH ₂ PO₄, 0.8M KH₂PO₄, 0.1M NaHEPES pH 7.5 at a protein to precipitant ratio of 1:2. A crystal was cryo-protected using well solution supplemented with 30% (v/v) glycerol and flash-cooled in liquid nitrogen.

Data Collection: Resolution (scaled): 1.75 Å; X-ray source: Synchrotron - Diamond I04.