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Materials and Methods: PvGGPPS (2IHI)
| Structure | Pv-FDPS: Plasmodium vivax farnesyl diphosphate synthase |
|---|---|
| PDB Code | 2IHI |
| 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 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 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 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 oC. |
| Protein stock concentration | 16.5 mg/mL for Pv-FDPS with His6-tag. |
| Crystallization | The native protein was crystallized by means by hanging drop vapor diffusion in a VDXm plate. The plate was set with 1.5 μL protein and 1.5 μL buffer in each drop, and 350 μL reservoir volume per well. Crystals grew overnight in 22% Peg 3350, 200 mM Li2SO4, 100 mM Tris, pH 8.5 at 18 ºC. |
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