Human Calpain 9

Target ID:

CAPN09

Aliases:

GC36nCL-4

Deposition Date:

Wednesday 27th April 2005

Families:

Proteases

Related Diseases:

SignallingCancer

Structure type:

apo

Download Coordinates:

1ZIV

Authors:

J.R.WALKER,T.DAVIS,E.M.NEWMAN,F.MACKENZIE,A.DONG,J.CHOE,C.ARROWSMITH,M.SUNDSTROM,A.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

1ZIV

tabs

Structure Details

After skin cancer, the most common cancer in the world is stomach cancer.
Furthermore, it has one of the worst five-year relative survival at about 15%.
The vast majority of these tumors are adenocarcinomas,
which develop from the innermost epithelial lining of the stomach\'s mucosa.
As this cancer grows slowly over many years, diagnosis has been difficult;
hence, treatment is limited to surgery (gastrectomy) and chemotherapy with the relatively unspecific anticancer drug, 5-fluorouracil.
The cause of gastric cancer is unknown, but the following risk factors have been shown to be important:
Helicobacter pylori infection, diet (salted, cured and smoked foods), tobacco and alcohol, previous stomach surgery, pernicious anemia, gender, age, and genetic factors.

At the molecular level, gastric cancer has been correlated with dysregulated activity of two gene products: p53 and calpain-9.[1]
In response to DNA damage, the transcription factor, p53 upregulates the expression of numerous proteins that are involved with either cell-cycle-arrest or apoptosis,
including the cyclin-dependent kinase inhibitor p21 and the BCL-related BID and BAX proteins.

Materials and Methods

CAPN9

PDB:1ZIV

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC027993
Entry Clone Source:MGC
SGC Clone Accession:capn09.028.347:D1 1ziv 02D01
Tag:N-terminal histag with integrated thrombin cleavage site: mgsshhhhhhssglvpr*gs
Host:BL21(DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsSFEQMRQECLQRGTLFEDADFPASNSSLFYSERPQIPFVWKRPGEIVKNPEFILGGATRTDICQGELGDCWLLAAIASLTLNQKALARVIPQDQSFGPGYAGIFHFQFWQHSEWLDVVIDDRLPTFRDRLVFLHSADHNEFWSALLEKAYAKLNGSYEALKGGSAIEAMEDFTGGVAETFQTKEAPENFYEILEKALKRGSLLGCFIDTRSAAESEARTPFGLIKGHAYSVTGIDQVSFRGQRIELIRIRNPWGQVEWNGSWSDSSPEWRSVGPAEQKRLCHTALDDGEFWMAFKDFKAHFDKVEICNLTPDALEEDAIHKWEVTVHQ
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:Using the SGC's LEX bubbling system, CAPN9 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

Extraction

Procedure
Frozen 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 (50 mM Tris pH 8.0 (VWR EM-9210), 500 mM NaCl (VWR EM-SX0420-1), 1mM EDTA), 1mM phenylmethanesulfonyl fluoride (Sigma P7626), 2mM CaCl2 and 1mL Sigma general protease inhibitor (Sigma P2714-1BTL, resuspended according to manufacturer's 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:
Ligand
MassSpec:
Crystallization:Purified CAPN9 was crystallized using the sitting drop vapor diffusion method. Diffracting crystals leading to the structure grew when the protein was mixed at 20 mg/mL with the reservoir solution (containing 18% Peg3350, 0.1M hepes pH 7.7, 0.2M CaCl₂) in a 1:1 volume ratio.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Davis TL, Walker JR, Finerty PJ Jr, Mackenzie F, Newman EM, Dhe-Paganon S. The crystal structures of human calpains 1 and 9 imply diverse mechanisms of action and auto-inhibition. J Mol Biol. 2007 Feb 9;366(1):216-29.