FKBP8
PDB:2AWG
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_036313
Entry Clone Source:MGC
SGC Clone Accession:fkbp08.090.206:H3; SGC031 H3
Tag:His-tag with integrated thrombin-cleavage site MGSSHHHHHHSSGLVPR*GS.
Host:E.coliBL21 (DE3)
Construct
Prelude:Sequence:gsPEEWLDILGNGLLRKKTLVPGPPGSSRPVKGQVVTVHLQTSLENGTRVQEEPELVFTLGDCDVIQALDLSVPLMDVGETAMVTADSKYCYGPQGRSPYIPPHAALCLEVTLKTAVD
Vector:p28a-LIC
Growth
Medium:TB
Antibiotics:Procedure:FKBP8 was expressed in E. coli BL21 (DE3) grown in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin at 37ºC to an OD600 of 7.5. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 0.05 mM, and incubated overnight at 15ºC. The culture was centrifuged and the cell pellets were collected and stored at -80ºC.
Purification
ProcedureIMAC purification: 4 microL of clarified supernatant is reserved for later analysis by SDS-PAGE. The rest of the clarified supernatant is then diluted 1:2 in lysis buffer, and loaded at approximately 1mL/min by gravity onto 5 mL of Ni-NTA resin (Qiagen 30450). 5 column volumes of lysis buffer are used to wash the column at approximately 3 mL/min, followed by 5 column volumes of low imidazole buffer (lysis buffer + 10 mM Imidazole (VWR EM-5720) pH 8) at approximately 3 mL/min. A 4 ?L sample of the low imidazole wash is saved for later analysis by SDS-PAGE. Samples are eluted from the Ni-NTA resin by exposure to 10 mL elution buffer (lysis buffer + 250 mM imidazole and 10% glycerol (EMD GX0185-5)) at 1mL/min flow rate. A 10 ?L sample of the eluate is saved for SDS-PAGE analysis. 10 ?L of each eluate is saved for measurement of protein concentration using Bradford reagent (BioRad 500-0202).
(His)6-tag cleavage: The 10 mL of Ni-NTA eluate from 2 is divided into 2x5 mL samples in 50 mL conical vials (352096, BD Biosciences). 5 mL of uncut sample is loaded onto gel filtration (see below); 1 unit of thrombin (Sigma T9681) per milligram of protein is added to the other 5 mL sample. The conical vial is stored without shaking, overnight, at 4ºC.
Size exclusion chromatography: All gel filtration columns, buffers, and protocols are identical for uncut and thrombin-treated proteins. A an XK 16x65 column (part numbers 18-1031-47 and 18-6488-01, GE Healthcare) packed with HighLoad Superdex 200 resin (10-1043-04, GE Healthcare) is pre-equilibrated with gel filtration buffer (lysis buffer + 5 mM ?-ME (Sigma 63689)) for 1.5 column volumes using an AKTAxpress (18-6645-05, GE Healthcare) at a flow rate of 3 mL/min. 5 mL of sample is loaded onto the column at 1.5 mL/min, and 2mL fractions are collected into 96-well plates (VWR 40002-012) using peak fractionation protocols with the following parameters: (Slope; min. peak width 0.833 min; level 0.000 mAU; peak start slope 10.000 AU/min; peak end slope 20.000 AU/min). Peak fractions are analyzed for purity using SDS-PAGE or visual analysis of the chromatogram and pooled.
Concentration: Purified proteins are concentrated using either 4 mL or 15 mL concentrators with an appropriate molecular weight cut-off (Amicon Ultra-15 10,000 MWCO, UFC901024 or 5,000 MWCO, UFC900524, as appropriate, Millipore) to a final concentration of 20 mg/mL for crystallographic screening or other biophysical studies.
Extraction
ProcedureFrozen cell pellets contained in bags (Beckman 369256) obtained from 2L liters of culture are thawed by soaking in warm water for 5 minutes. Each cell pellet is resuspended in 20 mL lysis buffer + 1mM phenylmethanesulfonyl fluoride (Sigma P7626), and 1mL Sigma general protease inhibitor (Sigma P2714-1BTL), resuspended according to manufacturerÂs instructions) and then homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis is accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol is 10 sec pulse at half-maximal frequency (5.0), 10 second rest, for 6 minutes total sonication time per pellet. Lysed cells are placed into centrifuge tubes (363647, Beckman Coulter) and centrifuged in a JA25.50 rotor in an Avanti J-20 XPI centrifuge (Beckman Coulter) for 20 minutes at 69,673 x g. The supernatant is decanted into a beaker, and the insoluble pellet discarded.
Concentration:LigandMassSpec:Crystallization:Purified FKBP8 was crystallized using the hanging drop vapor diffusion method. Crystals grew when the protein was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 0.5M NHSO
4, 1M LiSO
4, 0.1M Na-Citrate at pH 5.6 in 293K temperature, with 15% glycerol added as cryo protectant.
NMR Spectroscopy:Data Collection:Data Processing: