Useful Information

Structure Description

Please contact us for any questions or request for reagents.

Materials and Methods: PvGGPPS (3EZ3)

Structure	PVX_092040
PDB Code	3EZ3
Entry clone accession	Pv092040
Entry clone source	Plasmodium vivax Salvador I genomic DNA
SGC clone accession	Pv-PF11_0295; plate MAC01Q:A12
Tag	N-terminal: His6-tag with integrated TEV protease site: mhhhhhhssgrenlyfq*g
Construct sequence	mgsshhhhhhssgrenlyfqgMKETNSEEADSGLAFFRNMYDKYRDAFLSHLNEYSLEEEIKEHISKYYKLLFDYNCLGGKNNRGILVILIYEYVKNRDINSSEWEKAACLAWCIEILQAAFLVADD IMDKGEMRRNKYCWYLLKDVETKNAVNDVLLLYNSIYKLIEIYLRNESCYVDVIATFRDATLKTIIGQHLDTNIFSDKYSDAHREIDVNNINVPEQPVIDINMINFGVYKNIVIHKTAYYSFFLPIV CGMLLAGIAVDNLIYKKIEDISMLMGEYFQIHDDYLDIFGDSTKTGKVGSDIQNNKLTWPLIKTFELCSEPDKIKIVKNYGKNNLACVKVIDSLYEQYKIRKHYESYEKAQKAKILSAINELHHEGI EYVLKYLLEILFTGV
Vector	p15-tev-lic
Expression host	E. coli BL21-(DE3)-R3-pRARE2
Growth medium	TB
Growth method	A single colony was inoculated into 10 mL of LB with of Antibiotics and incubated with shaking at 250 rpm overnight at 37 ºC. The culture was transferred into 50 mL of TB with Antibiotics in a 250 mL shaking flask and incubated at 37 ºC for 3 hours. The culture was then transferred into 1.8 L of above-specified growth medium with Antibiotics 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.
Extraction buffers	Binding Buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, and 5 % glycerol
Extraction procedure	Cells were resuspended to approximately 40 mL/L of cell culture in Binding Buffer 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.
Purification buffers	Wash Buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM imidazole, 5 % glycerol Elution Buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 250 mM imidazole, and 5 % glycerol
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 at 2 – 2.5 mL/min. After washing, the protein was eluted with 15 mL of Elution Buffer. 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 sample was loaded onto a Sephadex S200 26/60 gel filtration column pre-equilibrated with 10 mM HEPES, pH 7.5 and 500 mM NaCl. The collected fractions corresponding to the correct eluted protein peak were concentrated using a 15 mL Amicon Ultra centrifugal filter device (Millipore). The protein sample identity and purity were evalulated by mass spectroscopy and SDS-PAGE gel. The concentrated sample was stored at 4 °C.
Protein stock concentration	11.2 mg/mL
Crystallization	1.5 μL of protein at 11.2 mg/mL containing an additional 10 mM ZOL, 10 mM IPP and 10 mM MgCl₂ was mixed with 1.5 μL of reservoir solution (20% PEG 3350, 200 mM Li2SO₄, 100 mM Tris, 8.5 at 18 °C) and incubated in hanging drops over 350 μL of reservoir solution. Crystals appeared overnight.