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Horizontal Tabs
Experimental Procedures
JMJD1A
Expression
Media:
Virus amplification: Sf900 III (Gibco) + 2% FBS.
Expression: Insect-Xpress (Lonza).
Expression protocol:
0.6L of Sf9 insect cells in 3L glass non-baffled flasks was infected with 1.8mL of virus P2 (expression can be performed in 1L of cells but the yield calculated per 1L is higher when performed in 0.6L). Cell density at infection time: 2e6 /mL. Protein was expressed for 72h at 27oC with 100rpm shaking.
Extraction
Extraction buffers:
Lysis buffer: 50mM HEPES-KOH, 0.3M KCl, 5% glycerol, 10mM imidazole pH 7.4, protease inhibitors cocktail set VII (Calbiochem)
Extraction procedure:
Cells were spun at 900xg for 10min and resuspended in 30 mL of lysis buffer per litre. Cells were broken by sonication (3min, 35% amplitude, 5s on 10s off). Insoluble fraction removed by centrifugation for 30 minutes at 55 000xg.
Purification
Column 1
IMAC: Sepharose 6 FF resin charged with nickel (GE/Amersham Biosciences).
Lysis: 50mM HEPES, 0.3 KCl, 5% glycerol, 10mM imidazole, pH 7.5
Wash: 50mM HEPES, 0.3M KCl, 5% glycerol, 40mM imidazole, pH 7.5
Elution: 50mM HEPES, 0.3M KCl, 5% glycerol, 250mM imidazole, pH 7.5 + protease inhibitors cocktail set VII (Calbiochem),
Procedure:
Proteins were batch bound to 0.5mL resin for one hour at 4OC. Resin was spun at 500 x g and supernatant removed. Resin was re-dissolved in 20 column volumes of binding buffer and centrifuged again. Resin was again re-dissolved in 10 CV of wash buffer and transferred to gravity column. Proteins were eluted with 5CV of elution buffer. Fractions containing proteins were analysed on SDS-page and pooled together.
Column 2:
Gel Filtration: HiLoad 16/60 Superdex 200 prep grade, 120 mL (GE/ Amersham Biosciences).
Buffer: 20mM Tris-HCl, 0.5M NaCl, 5% glycerol pH 7.6
Procedure:
Pooled fractions were filtered and loaded on gel filtration column (flow 1.2ml/min). Fractions corresponding to monomeric protein were pooled together.
Concentration and storage
The protein was concentrated using an Amicon Ultracel centrifugal concentrator (50 kDa MWCO) to 2 mg/ml by A280 and extinction coefficient. Aliquots were frozen in liquid nitrogen and kept in -80oC.
Yield
1.9mg from 0.6L
JMJD1B
Expression
Media:
Virus amplification: Sf900 III (Gibco) + 2% FBS.
Expression: Insect-Xpress (Lonza).
Expression protocol:
4x1L of Sf9 insect cells in 3L glass non-baffled flasks was infected with 3mL of virus P2. Cell density at infection time: 2e6 /mL. Protein was expressed for 72h at 27oC with 100rpm shaking.
Extraction
Extraction buffers:
Lysis buffer: 50mM HEPES-KOH, 0.3M KCl, 5% glycerol, 10mM imidazole pH 7.4, protease inhibitors cocktail set VII (Calbiochem)
Extraction procedure:
Cells were spun at 900xg for 10min and resuspended in 30 mL of lysis buffer per litre. Cells were broken by sonication (3x3min, 35% amplitude, 5s on 10s off). Insoluble fraction removed by centrifugation for 30 minutes at 55 000xg.
Purification
Column 1
IMAC: Sepharose 6 FF resin charged with nickel (GE/Amersham Biosciences).
Lysis: 50mM HEPES, 0.3 KCl, 5% glycerol, 10mM imidazole, pH 7.5
Wash: 50mM HEPES, 0.3M KCl, 5% glycerol, 40mM imidazole, pH 7.5
Elution: 50mM HEPES, 0.3M KCl, 5% glycerol, 250mM imidazole, pH 7.5 + protease inhibitors cocktail set VII (Calbiochem),
Procedure:
Proteins were batch bound to 3mL resin for one hour at 4OC with gentle rotation. Resin was spun at 500 x g and supernatant removed. Resin was re-dissolved in 60 column volumes of binding buffer and centrifuged again. Resin was again re-dissolved in 10 CV of wash buffer and transferred to gravity column. Proteins were eluted with 5CV of elution buffer. Fractions containing proteins were analysed on SDS-page and pooled together.
Column 2
Gel Filtration: HiLoad 16/60 Superdex 200 prep grade, 120 mL (GE/ Amersham Biosciences).
Buffer: 20mM HEPES, 0.3M KCl, 5% glycerol pH 7.6, 0.5mM TCEP
Procedure:
Pooled fractions were filtered and loaded on 2x gel filtration column (flow 1.2ml/min). Fractions corresponding to monomeric protein were pooled together (it’s impossible to separate some dimer-monomer fraction of the peak).
Concentration and storage
The protein was concentrated using an Amicon Ultracel centrifugal concentrator (50 kDa MWCO) to 1.3 mg/ml by A280 and extinction coefficient. Aliquots were frozen in liquid nitrogen and kept in -80oC.
Mutant protein for Crystallography and Crystallisation Conditions
Protocols for JMJD1BA-c078 (Q1601H:G1606R)
1. Protein expression and purification
Vector: pNIC28-Bsa4
Cell line: BL21[DE3]-pRARE2
Tags and additions: N-terminal, TEV protease cleavable hexahistidine tag
Final protein sequence
MHHHHHHSSGVDLGTENLYFQ*SMTSHSWLCDGRLLCLHDPSNKNNWKIFRECWKQGQPVLVSGVHKKLKSELWKPEAFSQEFGDQDVDLVNCRNCAIISDVKVRDFWDGFEIICKRLRSEDGQPMVLKLKDWPPGEDFRDMMPTRFEDLMENLPLPEYTKRDGRLNLASRLPSYFVRPDLGPKMYNAYGLITAEDRRVGTTNLHLDVSDAVNVMVYVGIPIGEGAHDEEVLKTIDEGDADEVTKERIHDHKEKPGALWHIYAAKDAEKIRELLRKVGEEQGQENPPDHDPIHDQSWYLDQTLRKRLYEEYGVQGWAIVQFLGDAVFIPAGAPHQVHNLYSCIKVAEDFVSPEHVKHCFRLTQEFRHLSNTHT
(underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
Cell Lysis
Lysis Buffer: 10 mM HEPES, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP, 10 mM imidazole pH 7.5
Cell pellet (180 g) was dissolved in 500 mL of lysis buffer and lysozyme and benzonase were added to 0.5 mg/mL and 1 μg/mL respectively. After 30 minutes stirring in the cold room 30 mL of 10 % Triton X-100 was added and stirring continued for a further 30 minutes. Cells were split across 24 x 50 mL Falcoln tubes and frozen at -20 °C overnight. Cells were thawed at room temperature and the volume of each tube brought to 50 mL with Lysis Buffer. Cells were centrifuged for 1 h at 5,000 g (4 °C).
Column 1: His GraviTrap columns (24 x 1 ml volume in a gravity-flow column)
Wash Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 10 mM imidazole, 0.5 mM TCEP
Elution Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 500 mM imidazole, 0.5 mM TCEP
The clarified cell extract was added to a 24 x 1 ml His GraviTrap columns pre-equilibrated with Wash Buffer. The columns were then washed with 2 x 5 ml Wash Buffer. The protein was eluted with 2.5 ml Elution Buffer.
Column 2: PD-10 desalting columns (24 x 8.3 ml volume in a gravity-flow column)
Wash Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 10 mM imidazole, 0.5 mM TCEP
Each 2.5 mL His GraviTrap fraction (24 x) was applied directly to a PD-10 column (24 x) for desalting and eluted with 3.5 mL of Wash Buffer
Tag cleavage
1 mg of TEV protease was added to every 10 mg of the eluted protein and the digestion was performed overnight at 4 °C.
Column 3: His GraviTrap columns (24 x 1 ml volume in a gravity-flow column)
Wash Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 10 mM imidazole, 0.5 mM TCEP
The TEV cleaved protein (3.5 mL) was applied to a His GraviTrap column pre-equilibrated in Wash Buffer for removal of His-tag, TEV and uncleaved protein. The column was washed with a further 2.5 mL of Wash Buffer for a final pool of 6 mL. The various desalted protein fractions were combined (24 x 6 mL) and 16 mL of 1 M L-arginine + 1 M L-glutamate was added to help avoid precipitation. The protein was concentrated to 25 mg/mL using a 30 kDa MWCO concentrator.
Column 4: Yarra SEC 2000 300x21.2 mm column (300x21.2 mm column, 104 mL volume)
Gel Filtration Buffer: 10 mM HEPES pH 7, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP
The column was equilibrated with Gel Filtration buffer and the protein loaded, the peak corresponding to JMJD1BA was taken and concentrated to 25 mg/ml using a 30 kDa MWCO concentrator, flash frozen in liquid nitrogen and stored at -80°C.
Protein crystallization
JMDJD1BA was crystallized by mixing 100 nl of 25 mg/ml protein in 10 mM HEPES pH7.5, 500mM NaCl, 5% Glycerol, 0.5 mM TCEP with 50nl of reservoir solution containing 25 % PEG3350, 200 mM MgCl2, 0.1 M bis-tris, pH 6.5. Crystals appeared after 4-7 days from sitting drop plates at 4°C. JMJD1BA crystallized in space group P21 with unit cell dimensions of a = 58 Å, b = 94 Å, c = 93 Å, corresponding to two JMJD1BA molecules in the asymmetric unit. The crystals typically diffract between 1.6 and 1.8 Å.
Protocols for JMJD1CA-c095 (Q2495R)
1. Protein expression and purification
Vector: pNIC28-Bsa4
Cell line: BL21[DE3]-pRARE2
Tags and additions: N-terminal, TEV protease cleavable hexahistidine tag
Final protein sequence
MHHHHHHSSGVDLGTENLYFQ*SMIPHSWICEKHILWLKDYKNSSNWKLFKECWKQGQPAVVSGVHKKMNISLWKAESISLDFGDHQADLLNCKDSIISNANVKEFWDGFEEVSKRQKNKSGETVVLKLKDWPSGEDFKTMMPARYEDLLKSLPLPEYCNPEGKFNLASHLPGFFVRPDLGPRLCSAYGVVAAKDHDIGTTNLHIEVSDVVNILVYVGIAKGNGILSKAGILKKFEEEDLDDILRKRLKDSSEIPGALWHIYAGKDVDKIREFLQKISKEQGLEVLPEHDPIRDQSWYVNKKLRQRLLEEYGVRTCTLIQFLGDAIVLPAGALHQVQNFHSCIQVTEDFVSPEHLVESFHLTRELRLLKE
(underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
Cell Lysis
Lysis Buffer: 10 mM HEPES, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP, 10 mM imidazole pH 7.5
Cell pellet (50 g) was dissolved in 200 mL of lysis buffer and lysozyme and benzonase added to 0.5 mg/mL and 1 μg/mL respectively. After 30 minutes stirring in the cold room, 30 mL of 10 % Triton X-100 was added and stirring continued for a further 30 minutes. Cells were split across 6 x 50 mL Falcon tubes and frozen at -20 °C. Cells were thawed at room temperature and the volume of each tube brought to 50 mL with Lysis Buffer. Cells were centrifuged for 1 h at 5,000 g (4 °C).
Column 1: His GraviTrap columns (4 x 1 ml volume in a gravity-flow column)
Wash Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 10 mM imidazole, 0.5 mM TCEP
Elution Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 500 mM imidazole, 0.5 mM TCEP
The clarified cell extract was added to a 4 x 1 ml His GraviTrap columns pre-equilibrated with Wash Buffer.
The columns were then washed with 2 x 5 ml Wash Buffer. The protein was eluted with 2.5 ml Elution Buffer.
Column 2: PD-10 desalting columns (4 x 8.3 ml volume in a gravity-flow column)
Wash Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 10 mM imidazole, 0.5 mM TCEP
Each 2.5 mL His GraviTrap fraction (4 x) was applied directly to a PD-10 column (4 x) for desalting and eluted with 3.5 mL of Wash Buffer
Tag cleavage
1 mg of TEV protease was added to every 10 mg of the eluted protein and the digestion was performed overnight at 4 °C.
Column 3: His GraviTrap columns (4 x 1 ml volume in a gravity-flow column)
Wash Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 10 mM imidazole, 0.5 mM TCEP
The TEV cleaved protein (3.5 mL) was applied to a His GraviTrap column pre-equilibrated in Wash Buffer for removal of His-tag, TEV and uncleaved protein. The column was washed with a further 2.5 mL of Wash Buffer for a final pool of 6 mL. The various desalted protein fractions were combined (4 x 6 mL) and 3 mL of 1 M L-arginine + 1 M L-glutamate was added to help avoid precipitation. The protein was concentrated to 25 mg/mL using a 30 kDa MWCO concentrator.
Column 4: Yarra SEC 2000 300x21.2 mm column (300x21.2 mm column, 104 mL volume)
Gel Filtration Buffer: 10 mM HEPES pH 7, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP
The column was equilibrated with Gel Filtration buffer and the protein loaded, the peak corresponding to JMJD1CA was taken and concentrated to 32 mg/ml using a 30 kDa MWCO concentrator, flash frozen in liquid nitrogen and stored at -80°C.
2. Protein crystallization
JMDJD1CA was crystallized by mixing 100 nl of 32 mg/ml protein in 10 mM HEPES pH7.5, 500mM NaCl, 5% Glycerol, 0.5 mM TCEP with 50 nl of reservoir solution containing 25 % PEG6000, 10 % ethylene glycol, 10 mM ZnCl, 0.1 Mtris, pH 7.5. Crystals appeared after 2-4 days from sitting drop plates at 4°C. JMJD1CA crystallized in space group P 21 21 2 with unit cell dimensions of a = 111 Å, b = 165 Å, c = 46 Å, corresponding to two JMJD1CA molecules in the asymmetric unit. The crystals typically diffract between 1.7 and 1.9 Å.
Assay Conditions
Overview and Principle
The JMJD1A/B demethylase assay uses the peptide ARTKQTARK(2me)STGGKAPRKQLA-GGG Spacer-Biotin (Histone H3 Lys 9 di-methyl) as a substrate and relies on detection of product H3K9Me1-biotin bound to streptavidin donor beads by a rabbit polyclonal anti-H3K9Me1 antibody coupled to protein-A acceptor beads.
For both JMJD1A and JMJD1B, screens use low nM concentrations of enzyme (0.25 nM enzyme routinely used in the assay) and nM concentrations of peptide substrate (H3K9Me2 routinely used at 60 nM in assay screens). Assays are performed in 384-well proxiplates.
AlphaScreen Beads
Store at 4°C
H3K9Me2 substrate
H3K9Me2-biotin substrate is stored at -20°C in 24 ml aliquots at a concentration of 100 mM.
JMJD1A and JMJD1B Enzyme
JMJD1A and B are stored in 1 mM aliquots at -80°C and diluted to 100 nM stock for assays.
Protocol
- Calculate the amount of AlphaScreen beads required for the experiment.
- Prepare the required amount of beads in a dark box at 4X the final concentration in the order of addition: Antibody > Protein A Acceptor > Streptavidin Donor.
- Retrieve an aliquot of JMJD1A and 100 mM peptide from frozen and place on ice.
- Weigh out Ferrous Ammonium Sulphate (100 -150 mg), 2-Oxoglutarate (2 - 4 mg) and L-Ascorbic Acid (8 -16 mg) in 2.0 ml Eppendorf tubes and prepare the solutions as described in section 5.
- Compounds for routine screening at 100 mM top concentration in IC50s are stored at a concentration of 10 mM in Labcyte 384-polypropylene or 384-Low Dead Volume (LDV) plates. For IC50 determinations an eleven point IC50 of the compound is prepared using ECHO dose response software (see section 6.1 for a typical plate layout). For single point screening ECHO plate reformat software is used to prepare assay plates. A DMSO control is included on each assay plate (column 12) and 2, 4-PDCA (100 mM) is dispensed into each well of column 24 (100 nl transfer). Dry dispense 100 nl of the prepared compound IC50 dilution into a Proxiplate.
- Prepare JMJD1A/B enzyme at 0.5 nM and dispense 5 ml into columns 1 -24 of a 384-well proxiplates using a Multidrop Dispenser and seal the plate.
- Incubate the plate for 15 minutes on the bench at room temperature.
- Prepare enough substrate solution (Assay buffer containing 200 mM L-Ascorbic Acid, 20 mM FAS, 120 nM H3K9Me2 peptide, 10 mM a-ketoglutarate) and dispense 5 ml of substrate into columns 1 -24 of the assay plate. Seal the plate.
- Allow the enzyme reaction to proceed at room temperature for 5 minutes.
- Dispense across the plate 5 ml of assay buffer containing 30 mM EDTA, 800 mM NaCl to stop the reaction.
- In a dark box, dispense 5 ml of the AlphaScreen beads prepared in STEP 2 into every well of the assay plate. Seal the plate with an aluminium plate foil
- Incubate the plate for 120 minutes at room temperature.
- Read the plate on an AlphaScreen capable plate reader (BMG Labtech Pherastar FS or Perkin Elmer Envision).
Preparation of Solutions
Assay Buffer:
Prepare fresh every week and filter sterilize through a 0.2 micron filter: 50mM HEPES Ph 7.5, 0.01% Tween-20, 0.1% BSA. Store at 4°C
Ferrous Ammonium Sulphate (FAS):
Prepare FAS fresh each day. Make up 400 mM stock solution (156.856 mg/ml) in 20 mM HCl and then prepare 2 ml of 1 mM FAS in deionized H2O. Store at room temperature.
2-Oxoglutarate (2-OG):
Prepare 2-OG fresh each dat. Make up 10 mM stock solution in deionized H2O (1.901 mg/ml) and store at room temperature.
L-Ascorbic Acid (L-AA):
Prepare L-AA fresh each day. Make up 50 mM stock solution in deionized H2O (8.806 mg/ml) and store at room temperature.
Substrate Solution
|
|
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Volume Required For |
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|
Stock Concentration |
Working Concentration |
10 ml of Substrate |
20 ml of Substrate |
30 ml of Substrate |
L-Ascorbic Acid |
50 mM |
200 mM |
40 µl |
80 µl |
120 µl |
FAS |
1 mM |
20 mM |
200 µl |
400 µl |
600 µl |
H3K9Me2-Biotin |
100 mM |
0.12 mM |
12 µl |
24 µl |
36 µl |
2-OG |
10 mM |
10 mM |
10 µl |
20 µl |
30 µl |
Assay Buffer |
|
|
9738 µl |
19476 µl |
29214 µl |
Beads Detection Solution:
AlphaScreen donor beads and acceptor beads are mixed and pre-incubated with Anti-H3K9Me1 antibody for at least 1 hour before addition to the assay. Make up at 4X final concentration:
Streptavidin Donor 0.08 mg/ml Final in Assay = 0.02 mg/ml
Protein A Acceptor 0.08 mg/ml Final in Assay = 0.02 mg/ml
Anti-H3K9Me1 1.6 mg/ml Final in Assay = 0.2 mg/ml
Make up the beads in dark box in the order: Antibody > Protein A Acceptor > Streptavidin Donor. Cover in foil and keep at room temperature.
Compound and Assay Plate Layouts
Typical IC50 Plate Layout:
16 compounds are plated out per plate. Duplicate transfers are performed for each compound concentration. Final DMSO concentration must not exceed 1.0%.
ECHO dose response software is used to prepare each compound IC50 approximating to a 1:3 dilution for each compound with 11 concentration points. Maximum volume transferred is 100 nl.
- Ueda, J., Ho, J. C., Lee, K. L., Kitajima, S., Yang, H., Sun, W., Fukuhara, N., Zaiden, N., Chan, S. L., Tachibana, M., Shinkai, Y., Kato, H., and Poellinger, L. (2014) The hypoxia-inducible epigenetic regulators Jmjd1a and G9a provide a mechanistic link between angiogenesis and tumor growth. Molecular and cellular biology 34, 3702-3720
- Mimura, I., Nangaku, M., Kanki, Y., Tsutsumi, S., Inoue, T., Kohro, T., Yamamoto, S., Fujita, T., Shimamura, T., Suehiro, J., Taguchi, A., Kobayashi, M., Tanimura, K., Inagaki, T., Tanaka, T., Hamakubo, T., Sakai, J., Aburatani, H., Kodama, T., and Wada, Y. (2012) Dynamic change of chromatin conformation in response to hypoxia enhances the expression of GLUT3 (SLC2A3) by cooperative interaction of hypoxia-inducible factor 1 and KDM3A. Molecular and cellular biology 32, 3018-3032
- Cho, H. S., Toyokawa, G., Daigo, Y., Hayami, S., Masuda, K., Ikawa, N., Yamane, Y., Maejima, K., Tsunoda, T., Field, H. I., Kelly, J. D., Neal, D. E., Ponder, B. A., Maehara, Y., Nakamura, Y., and Hamamoto, R. (2012) The JmjC domain-containing histone demethylase KDM3A is a positive regulator of the G1/S transition in cancer cells via transcriptional regulation of the HOXA1 gene. International journal of cancer 131, E179-189
- Yamada, D., Kobayashi, S., Yamamoto, H., Tomimaru, Y., Noda, T., Uemura, M., Wada, H., Marubashi, S., Eguchi, H., Tanemura, M., Doki, Y., Mori, M., and Nagano, H. (2012) Role of the hypoxia-related gene, JMJD1A, in hepatocellular carcinoma: clinical impact on recurrence after hepatic resection. Annals of surgical oncology 19 Suppl 3, S355-364
- Ohguchi, H., Hideshima, T., Bhasin, M. K., Gorgun, G. T., Santo, L., Cea, M., Samur, M. K., Mimura, N., Suzuki, R., Tai, Y. T., Carrasco, R. D., Raje, N., Richardson, P. G., Munshi, N. C., Harigae, H., Sanda, T., Sakai, J., and Anderson, K. C. (2016) The KDM3A-KLF2-IRF4 axis maintains myeloma cell survival. Nature communications 7, 10258
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Slide 1. Fragment hits
Slide 2. Fragment hit interactions with C-terminal helix
Slide 3. Fragment hit disrupting C-terminal helix