Entry Clone Source: MGC

Entry Clone Accession: IMAGE:6480902

SGC Construct ID: DMPK1A-c026

GenBank GI number: gi|976144

Vector: pNIC28-Bsa4. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ]

Tags and additions: N-terminal, TEV cleavable hexahistidine tag

Final protein sequence:
sm QQLVLDPGFLGLEPLLDLLLGVHQELG
ASELAQDKYVADFLQWAEPIVVRLKEVRL
QRDDFEILKVIGRGAFSEVAVVKMKQTGQ
VYAMKIMNKWDMLKRGEVSCFREERDVLV
NGDRRWITQLHFAFQDENYLYLVMEYYVG
GDLLTLLSKFGERIPAEMARFYLAEIVMA
IDSVHRLGYVHRDIKPDNILLDRCGHIRL
ADFGSCLKLRADGTVRSLVAVGTPDYLSP
EILQAVGGGPGTGSYGPECDWWALGVFAY
EMFYGQTPFYADSTAETYGKIVHYKEHLS
LPLVDEGVPEEARDFIQRLLCPPETRLGR
GGAGDFRTHPFFFGLDWDGLRDSVPPFTP
DFEGATDTCNFDLVEDGLTAMVSGGGETL
SDIREGAPLGVHLPFVGYSYSCMALRDSE
VPGPTP

The N-terminal 2 residues, sm , derive from the vector, following TEV protease cleavage of the hexahistidine tag.

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

Transformation: The construct DNA was transformed into homemade chemically competent cells of the expression strain by a standard heat shock procedure.

Glycerol stock prepataion: A number of colonies from the transformation were used to innoculate 1 ml of LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day glycerol stocks were prepared from this overnight culture.

Expression: A glycerol stock was used to innoculate 10 ml of TB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day this starter culture was used to innoculate 1L of LB media containing 50 µg/ml kanamycin. After growth at 37°C until the OD ₆₀₀ was around 0.5, the temperature was reduced to 18°C. The cells were induced by the addition of 1.0 mM IPTG when the OD ₆₀₀ was around 0.8. The expression was continued overnight. Two of these 1L growths were combined for the subsequent protein purification.

Cell harvest: Cells were spun down, resuspended in Lysis Buffer, and frozen at -20°C.

Lysis Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 5 mM Imidazole pH 7.4, 0.5 mM TCEP

Cell Lysis: The resuspended cells were thawed and lysed by sonication. The cell debris was spun down.

Purification: The protein was purified by Ni-affinity chromatography, TEV protease digestion to remove the hexahistidine tag, and gel filtration.

Column 1: Ni-NTA (2 ml).

Column 1 Buffers: Binding Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 5 mM Imidazole pH 7.4, 0.5 mM TCEP; Elution Buffer: Binding buffer containing 30 mM, then 50 mM, then 250 mM Imidazole.

Column 1 Procedure: The clarified supernatent was passed through a column of DE52 resin to bind the DNA and then through a 2 ml volume Ni-NTA column. The Ni-NTA column was washed with Lysis Buffer, and then eluted with Binding Buffer containing 30 mM, then 50 mM, then 250 mM imidazole. The DMPK1A appeared in both the 50 mM and 250 mM imidazole elution fractions.

TEV protease digestion: The eluted fractions containing DMPK1A were pooled and TEV protease was added. The digestion was left overnight at 4°C. The progress of the reaction was checked by mass spectrometry.

Column 2: S200 16/60 Gel filtration.

Column 2 Buffers: GF buffer: 50 mM BisTrisPropane pH 6.5, 500 mM KCl, 0.5 mM TCEP

Column 2 Procedure: The GF column was pre-equilibrated with GF Buffer. The TEV protease digested Ni-NTA eluant was concentrated to a volume of about 8 ml. Two gel filtration runs were made, each loading half of the concentrated Ni-NTA eluant. The flow rate was 1.0 ml/min. Eluted proteins were collected in 1.8 ml fractions. The fractions containing protein were identified on a coomasie blue stained gel.

Concentration: The DMPK1A was exchanged into a buffer of 50 mM BisTrisPropane pH 6.5, 100 mM KCl, 0.5 mM TCEP and concentrated to 11 mg/ml (measured using a nanodrop machine) . The inhibitor BIM 8 (bisindolylmaleimide VIII, acetate salt) was added to a concentration of 0.5 mM.

Mass spec. characterisation: Measured: 46061; Expected: 46059

Crystallisation: Crystals grew from a 3:1 ratio of protein to precipitant solution (1M Ammonium Sulphate, 1.5% PEG3350, 0.1M BisTris pH 7.0), using the vapour diffusion method.

Data Collection: Crystals were cryo-protected by equilibration into precipitant solution containing 25% ethylene glycol, and then flash frozen in liquid nitrogen. Data was collected at the Swiss Light Source beamline X10. Resolution: 2.8 Å.