No. XX AAK1A PDB: 4WSQ 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 5 ml of the starter culture. Cultures were incubated at 37°C with shaking at 170 rpm until an OD600nm >= 2.0 was reached. The flasks were then cooled down to 18°C and 0.4mM IPTG added to induce protein expression overnight. 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 21.5K 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: 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 second, third, fourth and fifth 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 - S200 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: The Superdex S200 column was first equilibrated with Gel Filtration buffer. The protein fraction from above step was concentrated to <5ml using a centrifugal filter with a 10kDa 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: Size Exclusion Chromatography - S200 HiLoad 16/60 Superdex run on ÄKTA-Express Cation Exchange Chromatography. HiTrap SP HP 5ml column on ÄKTA-Express |
QA Buffer I: 50mM HEPES pH 7.5 |
Procedure: The HiTrap Q HP column was equilibrated with 50mL QA buffer I. The protein fraction from above step was concentrated to 5mL using a centrifugal filter with a 10kDa cut-off. The 5mL fraction was made up to 50ml with QA buffer I and applied onto the column. Bound protein was eluted in 0%-100% gradient with QB buffer II. Several peaks were found corresponding to different phosphorylation states of the protein. Peaks were pooled separately. |
Crystallization: Dephosphorylated protein was concentrated to 11 mg/mL. The compound K252a was added to a final concentration of 1.5 mM from a stock at 50 mM in 100% DMSO. Prior to setting up crystallization plates the solution of AAK1-K252a was incubated on ice for approximately 30 minutes. Sitting-drop vapour diffusion plates were prepared. Crystals grew under multiple conditions using freshly prepared protein. The best-diffracting crystals of the AAK1-K252a complex were obtained by mixing 50 nL of protein with 100 nL of a reservoir solution containing 20% PEG 3350, 0.5 mM Zinc Acetate. 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:2.1 Å resolutionX-ray source: Diamond Light Source, station I04, using monochromatic radiation at wavelength 0.92 Å Crystals belonged to spacegroup P212121 with unit cell parameters a=68.66 Å b=71.32 Å c=183.64 Å, α=90° β= 90 γ= 90°. Diffraction data were integrated and scaled using Mofslm and SCALA, respectively. Molecular replacement was used in Phaser MR and PDB entry 2buj 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.. |