Human membrane protein, palmitoylated 7, PDZ domain

PDB Code 3O46 Target Class Non-protein Kinase

Target MPP7
Alias n/a
Disease Area/Function cancer, signalling
Date Deposited 2010-07-26
Authors Nedyalkova L, Tong Y, Tempel W, Zhong N, Guan X, Landry R, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Park H

Struc Details Tabs

Structure Details
Structure Description in preparation
Materials & Methods
StructureMPP7
PDB Code3O46
Entry clone accession AT32-B7:BC038105.2
Entry clone source MGC
SGC clone accession HPC043-C04
Tag mgsshhhhhhssglvprgs Removed in the crystallized form
Construct comments MPP7:D135-P225
Construct sequencegsDSVKIIRLVKNREPLGATIKKDEQTGAIIVARIMRGGAADRSGLIHVGDELREVNGIPVEDKRPEEIIQILAQSQGAI
TFKIIPGSKEETP
Vector pET28a-LIC
Expression host BL21-V2R-pRARE2
Growth method LEX Bubbling. The Se-Met target protein was expressed in E. coli by inoculating 50 mL of overnight culture grown in Luria-Bertani medium into a 2 L of M9 salt medium in the presence of NIAAC, Thiamine and Vitamine B12 mix, fifteen mineral supplements, 0.5% glycerol, 50 mg/mL kanamycin and 35 mg/mL chloramphenicol at 37 degree. When OD600 reached ~1.2, the temperature of the medium was lowered to 18 degree. Then, the inhibitory amino acid cocktail (IAAC) and Se-Met mix were added to the culture and the culture was induced with IPTG at a 1 mM final concentration. The cells were allowed to grow overnight before harvested and flash frozen in liquid nitrogen and stored at -80 degree.
Extraction buffers50mM HEPES pH7.5, 500mM NaCl, 5mM Imidazole, 5% Glycerol, 5mM TCEP
Extraction procedure Frozen cells from 4L TB culture were thawed and resuspended in 150 mL extraction buffer with freshly added 0.5% CHAPS, and supplemented with protease inhibitor cocktail (SIGMA Catalog # P8849), and 3 uL benzonase (Sigma Catalog # E1014, 250U/uL), and lysed using microfluidizer at 15,000 PSI.
Purification buffersWashing Buffer: 50mM HEPES pH7.5, 500mM NaCl, 30 mM Imidazole, 5% Glycerol , 2 mM TCEPElution Buffer: 50mM HEPES pH7.5, 500mM NaCl, 300 mM Imidazole, 5% Glycerol, 2 mM TCEPGel filtration Buffer: 20mM HEPES pH7.5, 500mM NaCl, 5% Glycerol, 1mM TCEP)
Purification procedure The lysate was centrifuged at 15,000 rpm for 45 minutes and the supernatants were mixed with 8 mL 50% flurry of TALON Metal Affinity Resin and incubated at 4 degree on rotary shaker for one hour. The mixture was then centrifuged at 2500 rpm for 5 min and the supernant discarded. The beads were then washed with washing buffer, and finally the elution buffer. The flow-through was collected and further purifed by a Superdex-75 gel filtraton column pre-equilibrated with gel filtration buffer. Fractions containing the protein were collected and concentrated with Amicon Ultra-15 centrifugal filter. The purity of the preparation is tested by SDS-PAGE to be greater than 95%.
Protein stock concentration 12.4 mg/mL
Mass spec protein expected 10088, measured 10088
Crystallization Crystallization was setup using in situ proteolysis method in sitting drops with Red Wings and SGC-I screens initially. Diffracting crystals were found from initial screen plate for SGC A07.Crystal used for structure determination was grown in 30% PEG 1500, 0.2M NaCl, 0.1M HEPES buffer at pH 7.5. Crystals grow to a mountable size within 2 days
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