Entry Clone Source: Site directed mutagenesis |
Entry Clone Accession: n/a |
SGC Construct ID: VRK1A-c510 |
GenBank GI number: gi|4507903 |
Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Amplified construct sequence: Nucleotides in bold and underlined were multated from refseq in an attempt to reduce surface entropy in the translated protein.
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Final protein sequence (Tag sequence in lowercase): ^ TEV cleave site |
Tags and additions: Cleavable N-terminal His6 tag. |
Host: BL21 (DE3)R3-pRARE2 (Phage resistant strain). |
Growth medium, induction protocol: The expression plasmid was transformed into the host strain and plated on LB-agar containing 50µg/ml kanamycin and 35µg/ml chloramphenicol. Several colonies were combined to inoculate a 1ml culture in TB (+ 50µg/ml kanamycin, 35µg/ml chloramphenicol). The culture was grown overnight, glycerol was added to 15% v/v (from a 60% stock), and the resulting glycerol stock was frozen at -80°C in 100 µl aliquots. A loopful of cells from the glycerol stock was inoculated into 5ml of LB medium containing 100µg/ml kanamycin and 35µg/ml chloramphenicol and grown overnight at 37°C. Overnight culture was used to inoculate 1 liter of LB containing 50µg/ml kanamycin & 34µg/ml chloramphenicol. Culture was grown at 37°C until the OD600 reached ~3 and then cooled to 18°C for 1 hour. IPTG was added to 0.1 mM, and growth continued at 18°C overnight. The cells were collected by centrifugation then pellet re-suspended in 2x lysis buffer and frozen. |
Column 1: Ni-affinity, HisTrap Crude FF, 5 ml (GE Healthcare). |
Column 1 Buffers: |
Column 1 Procedure: The cell extract was loaded on the column at 4 ml/minute on an ÄKTA-express system (GE Healthcare). The column was washed with 10 volumes of lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 4 ml/min. The eluted peak of A280 was automatically collected. |
Column 2: Gel filtration, Hiload 16/60 Superdex S75 prep grade, 120 ml (GE Healthcare) |
Column 2 Buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 5% Glycerol, 0.5 mM TCEP. |
Column 2 Procedure: The eluted fractions from the Ni-affinity Histrap column were loaded on the gel filtration column in GF buffer at 1.2 ml/min. Eluted proteins were collected in 2-ml fractions and analyzed on SDS-PAGE. |
TEV protease digestion: Peak fractions containing VRK1 were pooled and TEV protease was added at a molar ratio of 1:30. The digestion was left overnight at 4°C. His-TEV and contaminating proteins were removed by passing to 300µl Ni resin pre-equilibrated in GF buffer. The flow through containing TEV-cleaved protein was collected, concentrated to 30mg/ml using a centricon centrifugal device with a 30kDa MWCO, frozen at -80°C in 500µl aliquots. |
Mass spectrometry characterization: Measured: 41078, 41163 (one phosphorylation site) and 41240 (2 phosphorylation sites). Expected mass of unmodified protein: 41076.2. |
Protein concentration: 2.5 mg of VRK1 was diluted into 15 ml of low salt buffer (5mM HEPES. pH 7.5, 200 mM NaCl, 1% glycerol). 2 molar excess of ligand was added to diluted VRK1 and protein-ligand complex was concentrated to 20 mg/ml using an Amicon 30 kDa cut-off concentrator. |
Crystallisation: Prior to crystallization, pure protein at 20 mg/ml was incubated with 1mM K00604a (ASC24, provided by the Shokat laboratory) compound. Optimized condition contained 0.2 M K/Na(tart) and 20% PEG 3350, pH 7.0. Viable crystals were obtained at 4°C in a sitting drop mixing protein and reservoir solution at 2:1 volume ratio. |
Data Collection: The crystals were cryo-protected with the mother liquor containing 25% ethylene glycol and flash frozen in liquid nitrogen. Diffraction data were collected at Diamond Beamline I02 using monochromatic radiation at wavelength 0.9790 Å. |