Human serologically defined colon cancer antigen 10 - cyclophilin-like domain

Target ID:

PPI70

Aliases:

NY-CO-10

Deposition Date:

Tuesday 18th July 2006

Families:

Isomerases

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2HQ6

Authors:

J.R.WALKER,T.DAVIS,R.PARAMANATHAN,E.M.NEWMAN,P.J.FINERTYJR.,F.MACKENZIE,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Toronto

2HQ6

tabs

Structure Details

Human SDCCAG10 is a 472 residue protein with an amino terminal cyclophilin domain and carboxy terminal region weakly similar to the ATP-binding cassette of SMC proteins (structural maintenance of chromosomes). Very little is known about the molecular function of this protein. Interestingly, it is specifically recognized as a colon cancer antigen by cytotoxic T lymphocytes (Scanlan et al., 1998). And, it appears to be overexpressed in DAOY cells, a permanent medulloblastoma cell line (Peyrl et al., 2003). Despite its poorly define biological role, SDCCAG10 may nevertheless be an attractive target for cancer therapy.

We have determined the 1.75 Angstrom resolution crystal structure of the cyclophilin domain of human SDCCAG10. Although the cyclophilin fold is conserved, none of the variable loops (B1-B2, A3-B8, B4-B5, and A1-B3) have conformations belonging to the PPIA group. Furthermore, active site residues of SDCCAG10 are relatively divergent, with unique residues at key catalytic positions.

Materials and Methods

SDCCAG10

PDB:2HQ6

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:MGC
SGC Clone Accession:ppi70.008.176:B6; plate SDC066 B6
Tag:
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsEPPTNGKVLLKTTAGDIDIELWSKEAPKACRNFIQLCLEAYYDNTIFHRVVPGFIVQGGDPTGTGSGGESIYGAPFKDEFHSRLRFNRRGLVAMANAGSHDNGSQFFFTLGRADELNNKHTIFGKVTGDTVYNMLRLSEVDIDDDERPHNPHKIKSCEVLFNPFDD
Vector:

Growth

Medium:
Antibiotics:
Procedure:Using the LEX bubbling system, the protein was expressed in E. coli BL21 (DE3) grown in Terrific Broth (TB) in the presence of 50 microG/mL of kanamycin at 3degºC 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 15degC. The culture was centrifuged and the cell pellets were collected and stored at -80degC.

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 (lysis buffer + 10 mM Imidazole (VWR EM-5720) pH 8) at approximately 3 mL/min. A 4 microL 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 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).

Extraction

Procedure
The cell pellet was resuspended in lysis buffer, inhibitor (0.1 microM phenylmethyl sulfonyl fluoride, PMSF) and lysed using Microfluidizer. The lysate was cleared by centrifugation.
Concentration:
Ligand
MassSpec:
Crystallization:Protein concentration: 20 mg/mL; 20% Peg 3350, 0.2M NaI; 50 mM Tris pH 8.0, 500 mM NaCl, 5 mM BME; hanging drop; 1+1; cryo: 20% glycerol; 298.0K
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Peyrl,A., Krapfenbauer,K., Slavc,I., Yang,J.W., Strobel,T., and Lubec,G. (2003). Protein profiles of medulloblastoma cell lines DAOY and D283: Identification of tumor-related proteins and principles. Proteomics 3, 1781-1800.

Scanlan,M.J., Chen,Y.T., Williamson,B., Gure,A.O., Stockert,E., Gordan,J.D., Tureci,O., Sahin,U., Pfreundschuh,M., and Old,L.J. (1998). Characterization of human colon cancer antigens recognized by autologous antibodies. International Journal of Cancer 76, 652-658.