PPIC

PDB:2ESL

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:4505991
Entry Clone Source:MGC
SGC Clone Accession:ppi66.025.212:D6; plate SDC023 D6
Tag:N-terminal His-tag with integrated thrombin-cleavage site MGSSHHHHHHSSGLVPRGS.
Host:E.coliBL21 (DE3)

Construct

Prelude:
Sequence:

mgsshhhhhhssglvprgsGAEGFRKRGPSVTAKVFFDVRIGDKDVGRIVIGLFGKVVPKTVENFVALATGEKGYGYKGSKFHRVIKDFMIQGGDITTGDGTGGVSIYGETFPDENFKLKHYGIGWVSMANAGPDTNGSQFFITLTKPTWLDGKHVVFGKVIDGMTVVHSIELQATDGHDRPLTNCSIINSGKIDVKTPFVVEIADW

Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:PPIC 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

Procedure
IMAC 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 at approximately 3 mL/min. A 4 µL 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 at 1mL/min flow rate. A 10 microL sample of the eluate is saved for SDS-PAGE analysis. 10 microL of each eluate is saved for measurement of protein concentration using Bradford reagent (BioRad 500-0202).

Size exclusion chromatography: 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 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 2 mL 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.

Extraction

Procedure
The cell pellet was resuspended in lysis buffer + protease inhibitor (0.1µM phenylmethyl sulfonyl fluoride, PMSF) and lysed using Microfluidizer. The lysate was cleared by centrifugation.
Concentration:15 mg/mL
Ligand
Cyclosporin AMassSpec:
Crystallization:Purified PPIC was crystallized using the hanging drop vapor diffusion method. Crystals grew when the protein (15 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 25% PEG MME 550, 0.1 M zinc acetate, 0.1 M MES at pH 6.5 in 293K temperature.
NMR Spectroscopy:
Data Collection:
Data Processing: