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Horizontal Tabs
PDB ID |
Structure Details |
Structure of PHIP bromodomain |
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PHIP bromodomain in complex with fragment |
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PHIP bromodomain in complex with fragment |
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PHIP bromodomain in complex with fragment |
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PHIP bromodomain in complex with fragment |
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PHIP bromodomain in complex with fragment |
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PHIP bromodomain in complex with fragment |
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PHIP bromodomain in complex with fragment |
1. Protein expression and purification
Vector: pNIC28-Bsa4
Cell line: BL21-Rosetta
Tags and additions: N-terminal, TEV protease cleavable hexahistidine tag
Final protein sequence
MHHHHHHSSGVDLGTENLYFQ*SMSYDIQAWKKQCEELLNLIFQCEDSEPFRQPVDLLEYPDYRDIIDTPMDFATVRETLEAGNYESPMELCKDVRLIFSNSKAYTPSKRSRIYSMSLRLSAFFEEHISSVLSDYKSALRFHKRNTITKR
(underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
Cell Lysis
Lysis Buffer: 50 mM Hepes pH 7.5, 500 mM NaCl, 5% Glycerol, 10 mM Imidazole pH 7.4, 0.5 mM TCEP, protease inhibitors (Sigma, set III)
Cell pellet was dissolved in lysis buffer and broken by 2 cycles of sonication for 6 minutes (3s on, 3s off). The cell debris was removed by centrifugation for 45 minutes at 35000 x g.
Column 1: Ni-Sepharose 6 FF (5 ml volume in a gravity-flow column)
Wash Buffer: 50 mM Hepes pH 7.5, 500 mM NaCl, 5% Glycerol, 10 mM Imidazole, 0.5 mM TCEP
Elution Buffer: 50 mM Hepes pH 7.5, 250 mM NaCl, 5% Glycerol, 250 mM Imidazole, 0.5 mM TCEP
The clarified cell extract was added to 5 ml of Ni-Sepharose 6 FF pre-equilibrated with Wash buffer and passed through a glass column. The column was then washed with 150ml Wash Buffer. The protein was eluted with 30ml Elution Buffer.
Tag cleavage
3mg of TEV protease was added to the eluted protein and the digestion was performed overnight at 4 °C.
Column 2: Superdex75 HiPrep Gel Filtration
GF Buffer: 50 mM Hepes pH 7.5, 500 mM NaCl, 5% Glycerol
Successful TEV cleavage was confirmed by mass spectrometry. The protein was concentrated and injected into a Superdex75 HiPrep column (pre-equilibrated in GF Buffer) at 1.2 ml/min. Eluted fractions where analysed by SDS-PAGE and only the purest samples where pooled for re-binding on a Ni-Sepharose column.
Column 3: Ni-Sepharose 6 FF (1 ml volume in a gravity-flow column)
The column was equilibrated with gel filtration buffer, the protein was applied and only the flow through was taken forward. The protein was concentrated to 13 mg/ml using a 10 kDa MWCO concentrator, flash frozen in liquid nitrogen and stored at -80°C.
2. Protein crystallization
PHIPA(2) was crystallized by mixing 100nl of 13mg/ml protein in 20mM HEPES pH7.5, 500mM NaCl, 5% Glycerol with 100nl of reservoir solution containing 0.1M HEPES pH 7.5, 0.15M Magnesium Chloride, 34% PEG3350. Crystals appeared overnight from sitting drop plates at 4°C. PHIP(2) crystallized in space group P21212 with unit cell dimensions of a=60Å, b=92Å, c=24Å, corresponding to one PHIP(2) molecule in the asymmetric unit. The crystals typically diffract between 1.5 and 1.8 Å.
3. PHIP AlphaScreen Assay
Introduction
This procedure is intended to measure the interaction between the second bromodomain of PHIP and a peptide that has acetylated lysine residues and can be used to assay compounds which bind to the bromodomains and inhibit this interaction. The assay uses a 6HIS tagged bromodomain protein and a biotinylated peptide in conjunction with the Ni-NTA AlphaScreen kit. The excitation of the donor bead (680 nm) causes the generation of short lived singlet oxygen which can only diffuse 200 nm in solution. When the donor and acceptor beads are in close proximity, by binding of the peptide by the PHIP, the singlet oxygen can transfer its energy to the acceptor bead resulting in a chemilluminescent signal.
A. Materials and Equipment
A.1 Equipment
- Access™ Laboratory Workstation fitted with Echo®-555 Acoustic Liquid Handler.
(Labcyte USA) - Multidrop® Combi Reagent Dispenser fitted with Small Tube, Plastic Tip Dispensing Cassette.
(Thermo Scientific, USA) - PHERAstar FS Plate reader
(BMG Labtech, Germany)
A.2 Consumables
- ProxiPlate-384 Plus, White 384-shallow well Microplate, 6008280.
(Perkin Elmer, USA) - Echo® Qualified 384-Well Polypropylene Microplate, Clear, Flat Bottom, P-05525.
(Labcyte, USA) - Echo® Qualified 384-Well COC Microplate, Low Dead Volume, Clear, Flat Bottom, LP-0200.
(Labcyte, USA) - Microplate lid, low profile, sterile, 656191.
(Greiner Bio-One, UK)
A.3 Chemicals and reagents
- AlphaScreen® Histidine (Nickel Chelate) Detection Kit, 6760619M. (Perkin Elmer, USA)
For the Assay buffer (25 mM HEPES pH7.4, 100mM NaCl, 0.1% BSA, 0.05% CHAPS)
- Gibco® Ultra-Pure HEPES (N-2-Hydroxyethylpiperazine-N-2-Ethane Sulfonic Acid), 11344-041.
(Life Technologies, USA) - CHAPS (3-[3-(Cholamidopropyl)dimethylammonio]-1-propanesulfonate), C-1019.
(AG Scientific, USA) - BSA (Bovine Serum Albumin) lyophilized powder, ≥98%, A7030.
(Sigma-Aldrich, USA) - NaCl (Sodium chloride) AnalaR NORMAPUR®, 27810.364.
(VWR, USA) - DMSO (Dimethyl sulfoxide) ReagentPlus®, ≥99.5%, D5879.
(Sigma-Aldrich, USA)
A.4 Protein and peptide
- HIS tagged PHIP Bromodomain protein, SGC: stored in aliquots at -80°C.
- Biotinylated Histone H4 tetra-acetylated Peptide:
SGRGK(ac)GGK(ac)GLGK(ac)GGAK(ac)RHRK(biotin), stored at -20°C in 10 µl aliquots at a concentration of 1 mM.
B Methods
B.1 Dose response
- B.1.1 Protein and peptide are prepared to 16 µM from which are prepared 14 1:2 serial dilutions in assay buffer plus one buffer only.
- B.1.2 Add 4 µl of assay buffer to all wells of columns 1-16 of a ProxiPlate-384 Plus assay plate, using the matrix multichannel.
- B.1.3 Add 4 µl of the protein dilution series to columns 1-16 of the assay plate using the matrix multichannel.
- B.1.4 Add 4 µl of the peptide dilution series to rows A-P of the assay plate using the matrix multichannel.
- B.1.5 Cover the plate and incubate at room temperature for 30 minutes.
- B.1.6 Prepare an Alpha bead suspension at 1:300 (15 µl of each bead type in 4500 µl assay buffer) under light restricted conditions.
- B.1.7 Add 8 µl of the bead suspension to columns 1-16 of the assay plate using the Multidrop combi.
- B.1.8 Cover the assay plate to protect it from light and incubate at room temperature for 1 hour.
- B.1.9 Taking care to protect from light, read the assay plate using the PHERAstar microplate reader.
B.2 Inhibition Assay
- B.2.1 Dispense compounds into assay plates using the Echo liquid handler. Into columns 12 and 24 dispense DMSO into alternate wells these will be the positive controls, alternate wells are used to monitor the effect of DMSO in the assay.
- B.2.2 Prepare a protein peptide mix that is :
375 nM protein and 50 nM peptide (225 nM and 50 nM final assay concentration) - B.2.3 Add 12 µl of the protein peptide mix to the assay plates using the Multidrop combi and incubate for 30 minutes at room temperature.
- B.2.4 Prepare Alpha beads at 1:300 in assay buffer and add 8 µl to the assay plates using the Multidrop combi under light restricted conditions, cover and incubate for 1 hour at room temperature.
- B.2.5 Taking care to protect from light, read the assay plate using the PHERAstar microplate reader.
- B.2.6 Calculate % inhibition values as follows
Determine IC50 values by plotting % inhibition vs compound concentration and fitting the data to a non-linear sigmoidal dose response equation (4 parameter fit).
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