Plasmodium yoelii Cyclophilin

Target ID:

PY00382

Deposition Date:

Tuesday 29th March 2005

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

1Z81

Authors:

A.MULICHAK,Z.ALAM,M.AMANI,J.LEW,G.WASNEY,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,A.BOCHKAREV,R.HUI,M.VEDADI,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

1Z81

tabs

Structure Details

Cyclophylins belong to the immunophilins family of proteins
and are identified based on their ability to bind to immunosuppressant drug,
cyclosporine A (CsA) and catalyzing the peptidyl-prolyl cis/trans isomerization.
Cyclosporin A is also a potent anti-malarial compound in vitro
and in vivo in mice.

Cyclophilins are widely distributed across species and expressed in most tissues.
They differ in their molecular mass and in their affinity for CsA,
although all have nanomolar dissociation constants.
CsA-cyclophilin complex binds to calcineurin
and inhibits its protein phosphatase activity
leading to blocking T-cell growth, resulting in immunosuppression.

PY00382,
cyclophilin from Plasmodium yoelii, is a 210 amino acid protein
with 82% amino acid sequence identity with PF08_0121,
Peptidyl-prolyl cis-trans isomerase (PPI) (EC 5.2.1.8) from Plasmodium falciparum.

Materials and Methods

Py-CyP: Plasmodium yoelii cyclophilin (PY00382)

PDB:1Z81

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PY00382
Entry Clone Source:Plasmodium yoelii 17NXL genomic DNA
SGC Clone Accession:PY00382:; plate 2001:E10
Tag:His-tag with integrated thrombin protease site: mgsshhhhhhssglvprgs
Host:E.coli BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsMKSNSKDSENKKVENLVLDDNDENTIIPYYLSNLLTNPSNPVVFMDINLGNNFLGKFKFELFQNIVPKTSENFRQFCTGEYKVNNLPVGYKNTIFHRVIKEFMIQGGDFINHNGSGSLSIYGEKFDDENFDIKHDKEGLLSMANSGPNTNGCQFFITTKKCEWLDGKNVVFGRIIDNDSLLLLKKIENVSVTPYIYKPKIPINVVECGEL
Vector:p28a-LIC

Growth

Medium:Terrific broth (TB)
Antibiotics:50 microG/mL kanamycin
Procedure:A single colony was inoculated into 10 mL of LB with of Antibiotics and incubated with shaking at 250 rpm overnight at 37 degC. The culture was transferred into 50 mL of above-specified medium with Antibiotics in a 250 mL shaking flask and incubated at 37 degC for 3 hours. The culture was then transferred into 1.8 L of above-specified growth medium with Antibiotics in a 2L baffled flask and grown at 37 degC overnight in an Innova shaker from New Brunswick Scientific (150 rpm).

Purification

Procedure
The cleared lysate was loaded onto a column of 3 mL Ni-NTA from Qiagen at 4 degC. The column was washed with 150 mL Wash Buffer and the protein was eluted with 15 mL Elution Buffer. About 10 mg of pure protein was obtained from 1L of cell culture. The purified protein was dialyzed overnight into Crystal Buffer at 4 degC and concentrated using a Amicon Ultra centrifugal filter device from Millipore (15 kD cutoff).

Extraction

Procedure
Cells were centrifuged and the cell pellets were resuspended in binding buffer with protease inhibitor (1 mM benzamidine-HCl and 1 mM phenylmethyl sulfonyl fluoride, PMSF) and flash frozen. The thawed cell pellet was lysed by a combination of 0.5% CHAPS (Sigma) and sonication (1x 30 sec). Lysate was cleared by centrifugation at 35,000 x g and passed through DE52 from Whatman in 0.5 M NaCl.
Concentration:15 mg/mL
Ligand
MassSpec:
Crystallization:The protein was crystallized by means by hanging drop vapor diffusion in a 24-well Linbro plate. The plate was set with 1.5 microL uncleaved protein (15 mg/mL) and 1.5 microL buffer in each drop, and 500 microL reservoir volume per well. Crystals emerged in 1.15 M ammonium sulfate, 200 mM lithium chloride, 100 mM Tris at pH 8.5 and 20 degC.
NMR Spectroscopy:
Data Collection:
Data Processing: