Entry Clone Source: MGC

Entry Clone Accession: IMAGE:4132071

SGC Construct ID: FDPSA-c003

GenBank GI number: gi|4503685

Vector: p11. Details [PDF]; Sequence [FASTA] or [GenBank]

Tags and additions: N-terminal tag: mgsshhhhhhssgrenlyfqghm

Protein Sequence (after tag removal):
GHMNGDQNSDVYAQEKQDFVQHFSQIVRV
LTEDEMGHPEIGDAIARLKEVLEYNAIGG
KYNRGLTVVVAFRELVEPRKQDADSLQRA
WTVGWCVELLQAFFLVADDIMDSSLTRRG
QICWYQKPGVGLDAINDANLLEACIYRLL
KLYCREQPYYLNLIELFLQSSYQTEIGQT
LDLLTAPQGNVDLVRFTEKRYKSIVKYKT
AFYSFYLPIAAAMYMAGIDGEKEHANAKK
ILLEMGEFFQIQDDYLDLFGDPSVTGKIG
TDIQDNKCSWLVVQCLQRATPEQYQILKE
NYGQKEAEKVARVKALYEELDLPAVFLQY
EEDSYSHIMALIEQYAAPLPPAVFLGLAR
KIYKRRK

Host : BL21(DE3)

Growth medium, induction protocol: Overnight cultures in LB (10 ml with100 µg/ml ampicillin) were used to inoculate 1 litre of LB medium containing 100 µg/ml ampicillin. Cultures were grown at 37°C until they reached an OD₆₀₀ of 0.6-0.8 and then induced with 1 mM IPTG. The temperature was adjusted to 18°C and expression was allowed to continue overnight. The cells were collected by centrifugation.

Extraction buffer, extraction method : The cell pellet was resuspended in 50 mM HEPES pH 7.5, 5 mM imidazole, 500 mM NaCl, 5% glycerol.and lysed using a high pressure cell disruptor. The lysate was centrifuged at 17,000 RPM for 30 minutes at 4°C and the supernatant was collected.

Column 1 : Ni-affinity, HisTrap, 1 ml (GE/Amersham)

Buffers: Binding: 50 mM HEPES pH 7.5, 5 mM imidazole, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP; Wash: 50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM imidazole, 5% glycerol, 0.5 mM TCEP ; Elution: 50 mM HEPES pH 7.5, 500 mM NaCl, 250 mM imidazole, 5% glycerol, 0.5 mM TCEP.

Procedure: The cell extract was loaded on the column at 1 ml/minute on an AKTA-express system (GE/Amersham). The column was then washed with 10 column volumes of Lysis buffer, 10 column volumes of wash buffer, and then eluted with elution buffer at 1 ml/min. The eluted peak at A₂₈₀ was automatically collected.

Column 2 : Hiload 16/60 Superdex 200 prep grade 120 ml

Buffers : 10 mM Hepes pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP.

Procedure: The eluted protein from the Ni-affinity HisTrap column was automatically loaded on the gel filtration column at 1 ml/min using an AKTA-express system. Eluted proteins were collected in 1 ml fractions

Enzymatic treatment : His-tagged TEV protease was added (50 µg per mg FDPS) and incubated at 4°C for 48 hours to remove the hexahistidine tag. The TEV protease and any uncleaved protein were removed by passing the solution over Ni-NTA agarose beads. The cleaved FDPS was concentrated to 16 mg/ml using a Vivaspin concentrator with 10 kD MW cutoff. Final protein buffer: 10 mM Hepes pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP.

Crystallisation: Zoledronate, isopentenyl pyrophosphate, and MgCl2 were prepared as 100 mM aqueous stock solutions and added to the protein to a final concentration of 2 mM each. Crystals were grown at 20°C in 300 nl sitting drops by mixing 150 nl of protein solution and 150 nl of precipitant consisting of 14% PEG 6000, 0.7 M LiCl, and 70 mM citrate pH 4.0.