Pv-2-cys-Prx


PDB:2H66

Revision


Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:Pv118545
Entry Clone Source:Plasmodium vivax Salvador I genomic DNA from CDC
SGC Clone Accession:
Tag:N-terminal: His6-tag with integrated thrombin protease site: mgsshhhhhhssglvpr*gs
Host:E. coli BL21-(DE3)-R3

Construct


Prelude:
Sequence: 
mgsshhhhhhssglvprgsPTYVGKEAPFFKAEAVFGDNSFGEVNLTQFIGKKYVLLYFYPLDFTFVCPSEIIALDKALDAFHERNVELLGCSVDSKYTHLAWKKTPLAKGGIGNIKHTLLSDITKSISKDYNVLFDDSVSLRAFVLIDMNGIVQHLLVNNLAIGRSVDEILRIIDAIQHHEKYGDVCPANWQKGKVSMKPSEEGVAQYLSTL

Vector:p28a-LIC

Growth


Medium:
Antibiotics:
Procedure:Pv-2CysPrx was expressed in E. coli BL21-(DE3)-Rosetta Oxford in Terrific Broth (TB) in the presence of kanamycin/chloramphenicol (50 microgram/mL and 25 microgram/mL respectively). A single colony was inoculated into 10 mL of LB with of kanamycin/chloramphenicol (50 microgram/mL and 25 microgram/mL respectively) in a 50 mL Falcon tube and incubated with shaking at 250 rpm overnight at 37 ºC. The culture was transferred into 50 mL of TB with 50 microgram/mL kanamycin in a 250 mL shaking flask and incubated at 37 ºC for 3 hours. Then the culture was transfer into 1.8 L of TB with 50 microgram/mL kanamycin and 0.3 mL of antifoam (Sigma) in a 2 L bottle and cultured using the LEX system to an OD600 of ~5, cooled to 15 ºC, and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 ºC.

Purification


Procedure
The cleared lysate was loaded onto a column prepacked with 10 g DE52 (Whatman) anion exchange resin (previously activated with 2.5 M NaCl and equilibrated with Binding Buffer) and subsequently onto a 1.0 – 2.5 mL Ni-NTA (Qiagen) column pre-equilibrated with Binding Buffer at approximately 1 – 1.5 mL/min. After the lysate was loaded, the DE52 was further washed with 20 mL of Binding Buffer. Each Ni-NTA column was then washed with 200 mL of Wash Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM imidazole, and 5 % glycerol) at 2 – 2.5 mL/min. After washing, the protein was eluted with 15 mL of Elution Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 250 mM imidazole, and 5 % glycerol). EDTA was immediately added to the elution fraction to 1 mM; and DTT was added to 1 – 5 mM after approximately 15 more minutes. The protein was dialyzed overnight at 4 oC against 10 mM HEPES, pH 7.5, 500 mM NaCl; and finally concentrated using a 15 mL Amicon Ultra centrifugal filter device (Millipore). The concentrated sample was stored at 4 degC.

Extraction


Procedure
The culture was harvested by centrifugation. Pellets from 4 L of culture were resuspended to approximately 40 mL/L of cell culture in Binding Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, and 5 % glycerol) with the addition of protease inhibitors (1 mM benzamidine and 1 mM phenylmethyl sulfonyl fluoride (PMSF)). Resuspended pellets stored at -80 oC were thawed overnight at 4 oC on the day before purification. Prior to mechanical lysis, each pellet from 1 L of culture was pretreated with 0.5 % CHAPS and 500 units of benzonase for 40 minutes at room temperature. Cells were mechanically lysed with a microfluidizer (Microfluidizer Processor, M-110EH) at approximately 18000 psi; and the cell lysate was centrifuged using a Beckman JA-25.50 rotor at ~75000 x g (24000 rpms) for 20 minutes at 10 oC.
Concentration:8 mg/mL
Ligand
MassSpec:
Crystallization:Purified Py-2CysPrx was crystallized using the hanging drop vapor diffusion method in a VDXm plate with 350 µL of mother liquor at 18 ºC. 1.5 µL of the protein solution was mixed with 1.5 µL of the reservoir solution containing 5% Peg 4K, 50 mM NaAc, 100 mM NaAc, pH 4.6. Crystals appeared overnight.
NMR Spectroscopy:
Data Collection:
Data Processing: