No. XX AAK1A PDB: 5L4Q Materials & Methods |
Entry Clone Source: MGC |
Entry Clone Accession: |
SGC Construct ID: AAK1A-c046 |
Coding DNA sequence: |
Expressed protein sequence: |
Vector: pNIC-CTH0 |
Tags and additions: AENLYFQSHHHHHH: C-terminal hexahistidine tag cleavable by TEV protease. |
Host: BL21 (DE3)-R3-pRARE2. Phage-resistant derivative of BL21 (DE3), with pRARE2 plasmid encoding rare codon tRNAs (chloramphenicol-resistant). |
Growth Medium & Induction Protocol: A glycerol stock was used to inoculate a 10ml starter culture containing LB media with 50µg/ml Kanamycin + 34 µg/ml Chloramphenicol . The starter culture was grown overnight at 37°C with shaking at 200 rpm. The following morning, flasks containing 1L TB/Kanamycin were each inoculated with 3 ml of the starter culture. Cultures were incubated at 37°C with shaking at 170 rpm until an OD600nm ⥠1.4 was reached. The flasks were then cooled down to 18°C and 0.4mM IPTG added to induce protein expression overnight at OD600nm ⥠2.0. Cells were harvested by centrifugation at 5000 rpm at 4°C for 15 min. Cell pellets from each flask were resuspended in 15ml Binding buffer (50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 5mM Imidazole; 0.5mM TCEP; 1:2000 Protease Inhibitor Cocktail) and frozen at -20°C. |
Extraction buffer, extraction method: The frozen cells were thawed. The cells were lysed by ultrasonication over 15 min with the sonicator pulsing ON for 5 sec and OFF for 10. A final concentration of 0.15% PEI was added to the lysate. The cell lysate was spun down by centrifugation at 22 K rpm at 4°C for 1 h. The supernatant was recovered for purification. |
Column 1: Ni-Affinity Chromatography. 5 ml of 50 % Ni-sepharose slurry was applied onto a 1.5 x 10 cm column. The column was equilibrated with binding buffer (50ml). |
Buffers: |
Procedure: Carried out in a cold room. The supernatant following centrifugation was applied by gravity flow onto the Ni-sepharose column. The bound protein was then washed with 50ml binding buffer and subsequently with 30 ml wash buffer. AAK1A protein was then eluted by applying a step gradient of imidazole - using 5 ml portions of elution buffer with increasing concentration of imidazole (1 x 50 mM, 1 x 100 mM, 1 x 150 mM and 2 x 250 mM). Fractions were analyzed by SDS PAGE and the first, second, third & fourth elution fractions were kept and pooled. |
Enzymatic treatment: TEV protease cleavage. Fractions containing AAK1A were treated with TEV protease overnight at 4°C. |
Column 2: Size Exclusion Chromatography - S75 HiLoad 16/60 Superdex run on ÄKTA-Express |
Gel Filtration buffer: 300 mM NaCl, 50 mM HEPES pH 7.5, 0.5 mM TCEP, pH 7.5, 5% glycerol |
Procedure: Column stored in a cold room. The Superdex S75 column was first equilibrated with Gel Filtration buffer. The protein fraction from above step was concentrated to <5ml using a centrifugal filter with a 30kDa cut-off, before being syringe injected onto the column through 0.2µM pore filter and eluted with Gel Filtration buffer. Fractions containing the target protein were pooled together. |
Column 3: Ni-Affinity Chromatography. 1 ml of 50 % Ni-sepharose slurry was applied onto a 1.0 x 10 cm column. The column was equilibrated with binding buffer (15ml). |
Buffers: |
Procedure: Carried out at room temperature, with fractions stored on ice immediately following collection. The pooled fractions from gel filtration were applied by gravity flow onto the Ni-sepharose column and flow-through collected. The column was then washed with 4ml wash buffer and subsequently with 5ml elution buffer. Fractions were analyzed by SDS PAGE and the flow-through containing AAK1A was used for crystallization. |
Crystallization: Dephosphorylated protein was concentrated to 12 mg/mL. The compound LKB1 (AAK1 dual inhibitor) was added to a final concentration of 1.5 mM from a stock at 50 mM in 100% DMSO (3% final DMSO concentration). Prior to setting up crystallization plates the solution of AAK1-LKB1 was incubated on ice for approximately 30 minutes, then centrifuged at 14,000 rpm for 10 minutes, 4°C. Sitting-drop vapour diffusion plates were prepared. Crystals grew under multiple conditions using freshly prepared protein. The best-diffracting crystals of the AAK1-LKB1 complex were obtained using a reservoir solution containing 26% PEG 3350, 0.1 M bis-Tris pH5.5 by spiking drops with 20 nL of seed-stock solution immediately prior to incubation at 18°C. Seed stock was prepared from poorly-formed crystals of AAK1-LKB1 grown during previous rounds of crystal optimisation, which were diluted in 50-100 µL reservoir solution and vortexed for 2 min in an Eppendorf containing a seed bead. A 1:1 dilution series of seeds was prepared in order to find the optimal seed concentration. Prior to mounting crystals were cryo-protected in situ by addition of reservoir solution containing an additional 25% ethylene glycol. Crystals were then flash frozen in liquid nitrogen. |
Data Collection:2 Å resolutionX-ray source: Diamond Light Source, station I02, using monochromatic radiation at wavelength 0.9795 Å Crystals belonged to spacegroup P1211 with unit cell parameters a=61.8 Å b=55.05 Å c=86.57 Å, α=90° β= 104.44 &= 90°. Diffraction data were integrated and scaled using xia2 & Aimless. Molecular replacement was used in Phaser MR and PDB entry 4wsq was used as a search model. Two molecules were present in the asymmetric unit. COOT, REFMAC and PHENIX.REFINE were used for model building and refinement. |