AKR7A2

PDB:2BP1

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:AKR 7A2A-s001
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal His-tag with TEV cleavage site: MHHHHHHSSGVDLGTENLYFQ*SH
Host:E.coli BL21 Rosetta

Construct

Prelude:
Sequence:

Vector:pLIC-SGC

Growth

Medium:
Antibiotics:
Procedure:The AKR 7A construct was expressed in E. coli (BL21 Rosetta) in 1 L Terrific Broth in the presence of 100 mg/mL of ampicillin and 34 mg/mL chloramphenicol at 37 ºC. Cells were induced at 1 mM IPTG as soon as the OD reached 0.6 and temperature was shifted to 25 ºC, and culture was grown for 12 hrs. Cells were collected by centrifugation and pellets were stored frozen (-20 ºC) until further use.

Purification

Procedure
Column 1 : HiTrap His. Buffers (adjusted to pH 8.0): Lysis buffer: 10mM Imidazole, 300mM NaCl, 50mM NaH 2 PO 4. Wash buffer: 20mM Imidazole, 300mM NaCl, 50mM NaH 2 PO 4. Elution Buffer: 250mM Imidazole, 300mM NaCl. Procedure: Sample was loaded, washed with wash buffer and eluted in elution buffer. The collected peak was injected into size-exclusion chromatography system, and the main peak was selected for concentration using an Amicon Ultra device.

Column 2 : Superdex S200. Buffers : 10 mM Hepes, pH 7.4, 500 mM NaCl, 5% glycerol.

Extraction

Procedure
Pellets were resuspended in 20 mL lysis buffer including Protease inhibitor (complete, Roche), lysed by French Press, nucleic acids were removed by 0.15% PEI precipitation, and the solution was centrifuged to obtain a clear supernatant (15 min, 20.000 x g). Supernatants were processed in a 2 step chromatographic procedure using the Akta xpress (GE Healthcare) purification system.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals in complex with NADPH were obtained using the following conditions: Vapour diffusion method, sitting drop, 293K, 0.2M Ammonium Citrate, 20% PEG 3350
NMR Spectroscopy:
Data Collection:
Data Processing: