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Horizontal Tabs
PDB ID |
Structure Details |
Compound ID |
Structure of GDP-bound Kalirin/RAC1 |
GT000001f | |
Kalirin/RAC1 in complex with fragment |
Z56880342 | |
Kalirin/RAC1 in complex with fragment |
SG000012 | |
Kalirin/RAC1 in complex with fragment |
SG000013 | |
Kalirin/RAC1 in complex with fragment |
SG000055 | |
Kalirin/RAC1 in complex with fragment |
SG000059 | |
Kalirin/RAC1 in complex with fragment |
SG000070 | |
Kalirin/RAC1 in complex with fragment |
SG000086 | |
Kalirin/RAC1 in complex with fragment |
SG000089 | |
Kalirin/RAC1 in complex with fragment |
SG000096 | |
Kalirin/RAC1 in complex with fragment |
SG000098 | |
Kalirin/RAC1 in complex with fragment |
SG000112 |
Protein expression and purification
Kalirin (1)
Vector: pNIC28-10His
Cell line: BL21(DE3)-R3-pRARE2
Tags and additions: N-terminal, TEV protease cleavable decahistidine tag
Final protein sequence:
MHHHHHHHHHHSSGVDLGTENLYFQ*SMRKKEFIMAELLQTEKAYVRDLHECLETYLWEMTSGVEEIPPGILNKEHIIFGNIQEIYDFHNNIFLKELEKYEQLPEDVGHCFVTWADKFQMYVTYCKNKPDSNQLILEHAGTFFDEIQQRHGLANSISSYLIKPVQRVTKYQLLLKELLTCCEEGKGELKDGLEVMLSVPKKANDAMHV
(underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
Protein Expression
A 10 mL overnight culture grown in LB containing kanamycin (final concentration 50 μg/mL) and chloramphenicol (34 μg/mL) at 37 °C was used to inoculate 1 L of AIM-TB (ForMedium) with 0.01% Antifoam 204 and antibiotics in a 2.5 L baffled flask (Ultrayield, Thomson). Cells were grown for 4 hours at 37 °C 250 rpm before the temperature reduced to 25 °C 250 rpm and shaken overnight. Cells were harvested by centrifugation at 4,000 x g for 20 minutes at 4 °C and the pellet stored at -20 °C.
Cell Lysis
Lysis Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 0.5 mM TCEP, 0.5 mg/mL Lysozyme, 0.1 μg/mL Benzonase and protease inhibitors (Calbiochem EDTA-free Protease Inhibitor Cocktail Set III)
The cell pellet was resuspended in lysis buffer (20 mL per 5g of pellet), Triton X-100 to a final concentration of 2% added and the cells frozen overnight at -80 °C. The cells were thawed in a room temperature water bath, imidazole added to a final concentration of 20 mM and the cell debris was removed by centrifugation for 1 hour at 4000 x g.
Column 1: His GraviTrap columns (3 x 1 mL volume of Ni-Sepharose 6 FF in a gravity-flow column)
Wash Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 20 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 3 x 1 mL of His GraviTrap columns pre-equilibrated with Wash buffer. The columns were then washed with 2 x 10 mL Wash Buffer. The protein was eluted with 2.5 mL Elution Buffer after applying column 1 directly onto column 2.
Column 2: PD-10 desalting columns (3 x 8.3 mL volume of Sephadex G-25 resin in a gravity-flow column)
Wash Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole, 0.5 mM TCEP
Following elution of the protein from the His GraviTrap column directly onto the PD-10 column, the HisGraviTrap column was removed and the protein eluted from the PD-10 column using 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 (3 x 1 mL volume of Ni-Sepharose 6 FF in a gravity-flow column)
Wash Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole, 0.5 mM TCEP
The TEV cleaved protein was applied to a HisGraviTrap column pre-equilibrated in Wash Buffer to remove TEV protease, His-tag and uncleaved protein. The columns were then washed with 2.5 mL of Wash Buffer for a final pool of 6 mL. Protein fractions were combined and concentrated to ~ 30 mg/mL using a 10 kDa MWCO concentrator.
Column 4: Yarra SEC 2000 300x21.2 mm column (300x21.2 mm column, 104 mL volume) (Gel filtration)
GF buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP
The protein was loaded onto the column pre-equilibrated in GF buffer, the peak corresponding to the target protein was taken, concentrated to 12 mg/ml and stored at -80 °C.
Kalirin (2)
Vector: pNIC28-Bsa4
Cell line: BL21(DE3)-R3-pRARE2
Tags and additions: N-terminal, TEV protease cleavable hexahistidine tag
Final protein sequence:
MHHHHHHSSGVDLGTENLYFQ*SMDREVKLRDANHEVNEEKRKSARKKEFIMAELLQTEKAYVRDLHECLETYLWEMTSGVEEIPPGILNKEHIIFGNIQEIYDFHNNIFLKELEKYEQLPEDVGHCFVTWADKFQMYVTYCKNKPDSNQLILEHAGTFFDEIQQRHGLANSISSYLIKPVQRVTKYQLLLKELLTCCEEGKGELKDGLEVMLSVPKKANDAMHVSMLEGFDENLDVQGELILQDAFQVWDPKSLIRKGRERHLFLFEISLVFSKEIKDSSGHTKYVYKNKLLTSELGVTEHVEGDPCKFALWSGRTPSSDNKTVLKASNIETKQEWIKNIREVIQERIIHLKGAL
(underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
Protein Expression
A 10 mL overnight culture grown in LB containing kanamycin (final concentration 50 μg/mL) and chloramphenicol (34 μg/mL) at 37 °C was used to inoculate 1 L of AIM-TB (ForMedium) with 0.01% Antifoam 204 and antibiotics in a 2.5 L baffled flask (Ultrayield, Thomson). Cells were grown for 4 hours at 37 °C 250 rpm before the temperature reduced to 25 °C 250 rpm and shaken overnight. Cells were harvested by centrifugation at 4,000 x g for 20 minutes at 4 °C and the pellet stored at -20 °C.
Cell Lysis
Lysis Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 0.5 mM TCEP, 0.5 mg/ml Lysozyme, 0.1 μg/ml Benzonase and protease inhibitors (Calbiochem EDTA-free Protease Inhibitor Cocktail Set III)
Cell pellet was resuspended in lysis buffer (20 mL per 5g of pellet), Triton X-100 to a final concentration of 2% added and the cells frozen overnight at -80 °C. The cells were thawed in a room-temperature water bath, imidazole added to a final concentration of 20 mM and the cell debris was removed by centrifugation for 1 hour at 4000 x g.
Column 1: His GraviTrap columns (3 x 1 mL volume of Ni-Sepharose 6 FF in a gravity-flow column)
Wash Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 20 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 3 x 1 mL of His GraviTrap columns pre-equilibrated with Wash buffer. The columns were then washed with 2 x 10 mL Wash Buffer. The protein was eluted with 2.5 mL Elution Buffer after applying column 1 directly onto column 2.
Column 2: PD-10 desalting columns (3 x 8.3 mL volume of Sephadex G-25 resin in a gravity-flow column)
Wash Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole, 0.5 mM TCEP
Following elution of the protein from the His GraviTrap column directly onto the PD-10 column, the HisGraviTrap column was removed and the protein eluted from the PD-10 column using 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 (3 x 1 mL volume of Ni-Sepharose 6 FF in a gravity-flow column)
Wash Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole, 0.5 mM TCEP
The TEV cleaved protein was applied to a HisGraviTrap column pre-equilibrated in Wash Buffer to remove TEV protease, His-tag and uncleaved protein. The columns were then washed with 2.5 mL of Wash Buffer for a final pool of 6 mL. Protein fractions were combined and concentrated to ~ 30 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)
GF buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP
The protein was loaded onto the column pre-equilibrated in GF buffer, the peak corresponding to the target protein was taken, concentrated to 20 mg/ml and stored at -80 °C.
RAC1
Vector: pNIC-NStIIT
Cell line: BL21(DE3)-R3-pRARE2
Tags and additions: N-terminal, TEV protease cleavable Strep-tag II
Final protein sequence:
HMSSGASWSHPQFEKGGGSGGGSGGAAWSHPQFEKGSGVDLGTENLYFQ*SMQAIKCVVVGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVMVDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHHCPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQRGLKTVFDEAIRAVL
(underlined sequence contains vector encoded Strep-tag and TEV protease cleavage site*)
Protein Expression
A 10 mL overnight culture grown in LB containing kanamycin (final concentration 50 μg/mL) and chloramphenicol (34 μg/mL) at 37 °C was used to inoculate 1 L of AIM-TB (ForMedium) with 0.01% Antifoam 204 and antibiotics in a 2.5 L baffled flask (Ultrayield, Thomson). Cells were grown for 4 hours at 37 °C 250 rpm before the temperature reduced to 25 °C 250 rpm and shaken overnight. Cells were harvested by centrifugation at 4,000 x g for 20 minutes at 4 °C and the pellet stored at -20 °C.
Cell Lysis
Lysis Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 0.5 mM TCEP, 0.5 mg/ml Lysozyme, 0.1 μg/ml Benzonase and protease inhibitors (Calbiochem EDTA-free Protease Inhibitor Cocktail Set III)
Cell pellet was resuspended in lysis buffer (20 mL per 5g of pellet), Triton X-100 to a final concentration of 2% added and the cells frozen overnight at -80 °C. The cells were thawed in a room temperature water bath and the cell debris was removed by centrifugation for 1 hour at 4000 x g.
Column 1: Strep-Tactin columns (3 x 1 mL volume of Strep-Tactin XT resin in a gravity-flow column)
Wash Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 0.5 mM TCEP
Elution Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 50 mM D-Biotin, 0.5 mM TCEP
The clarified cell extract was added to 3 x 1 mL of His GraviTrap columns pre-equilibrated with Wash buffer. The columns were then washed with 2 x 10 mL Wash Buffer. 2 mL of Elution Buffer was applied to each column. After 10 minutes and 20 minutes, this was repeated to result in a 6 mL pool of eluted protein.
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 2: His GraviTrap columns (3 x 1 mL volume of Ni-Sepharose 6 FF in a gravity-flow column)
Wash Buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole, 0.5 mM TCEP
The TEV cleaved protein was applied to a HisGraviTrap column pre-equilibrated in Wash Buffer to remove TEV protease. The columns were then washed with 2.5 mL of Wash Buffer. Protein fractions were combined and concentrated to ~ 30 mg/mL using a 10 kDa MWCO concentrator.
Column 3: Yarra SEC 2000 300x21.2 mm column (300x21.2 mm column, 104 mL volume) (Gel filtration)
GF buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP
The protein was loaded onto the column pre-equilibrated in GF buffer, the peak corresponding to the target protein was taken and concentrated to 15 mg/ml and stored at -80 °C.
Kalirin(1)/RAC1 GDP-free complex formation
GDP removal
HPLC buffer: 100 mM Potassium phosphate pH 6.5, 10 mM Tetrabutyl phosphonium bromide, 5% methanol
Equipment: Agilent 1200 series HPLC. Reverse-phase C-18 5 μM (250 x 4.6 mm)
10 x Exchange Buffer: 2M ammonium phosphate, 10 μM zinc chloride
Kalirin (1) and RAC1 were mixed in a 1:1 molar ratio for an hour and concentrated to 60 mg/mL (138 μL) using a 30 kDa MWCO concentrator. 40 μL of 10 mM β,γ-Methyleneguanosine 5’-triphosphate sodium salt (GppCp, Sigma) and 4 Units of Alkaline phosphatase (Roche) were added. 20 μL of the 10x exchange buffer was added (final concentration 1 x) and the mixture incubated at 4 °C for 3 hours (Final concentration of Kalirin(1)/RAC1 is 1 mM). Degradation of GDP to GMP and guanosine was monitored by HPLC. Following complete degradation of GDP, 0.008 Units of snake venom phosphodiesterase I (Sigma) was added. The reaction was left overnight at 4 °C and the degradation of GppCp to GMP and guanosine monitored by HPLC. The protein was diluted to 30 mg/mL, flash frozen in liquid nitrogen and stored at -80 °C.
Column 1: Yarra SEC 2000 300x21.2 mm column (300x21.2 mm column, 104 mL volume)
GF buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP
The protein was thawed and loaded onto the column pre-equilibrated in GF buffer, the peak corresponding to the complex taken, concentrated to 10.4 mg/mL and stored at -80 °C.
Protein crystallisation
Kalirin(1)/RAC1 was crystallised by mixing 75nL of 10.4 mg/mL protein in 10mM HEPES pH 7.5, 500mM NaCl, 5% Glycerol 0.5 mM TCEP with 75nL of reservoir solution containing 0.1M bis-tris pH 5.5, 24% PEG3350. Crystals appeared overnight from sitting drop plates at 20 °C. Kalirin(1)/RAC1 crystallised in space group P65 2 2 with unit cell dimensions of a=63 Å, b=63 Å, c=346 Å, corresponding to one Kalirin(1)/RAC1 molecule in the asymmetric unit. The crystals typically diffract between 1.7 and 2.3 Å.
Nucleotide exchange assay
Equipment
PHERAstar FSX Plate reader (BMG Labtech, Germany)
Consumables
Microplate, 384 well, PS, flat bottom, small volume, non-binding, black, 784900. (Grenier Bio-One, UK)
Chemicals and reagents
MgCl2 (magnesium chloride) M8266. (Sigma-Aldrich, UK)
GTP (guanosine 5'-triphosphate disodium salt trihydrate) JBS-NU-1012-100. (Enzo Life Sciences, UK)
Buffers
Dilution buffer (DB): 20 mM TRIS HCl pH7.5, 50 mM NaCl, 1 mM MgCl2, 0.1% BSA, 1 mM DTT
Exchange buffer (EB): 20 mM TRIS HCl pH7.5, 50 mM NaCl, 2 mM EDTA, 0.1% BSA, 1 mM DTT, 0.75 µM BODIPY™ FL GDP
Protein and peptide
RAC1 c001 (RAC1), SGC: stored in aliquots at -80°C.
Kalirin c002 (Kalirin (2)), SGC: stored in aliquots at -80°C.
RAC1 was prepared in a stock solution at 50 µM in DB, Kalirin (2) was prepared in stock solution at 1.25 µM in DB. Three vials were prepared: 2 µL of RAC1, 2 µL of Kalirin (2) and 63.5 µL of EB were mixed into Vial A; 2 µL of RAC1, 2 µL of Kalirin (2) and 63.5 µL of EB were mixed into Vial B; 4 µL of DB and 63.5 µL of EB were mixed into Vial C. The vials were incubated at room temperature for 20 minutes, then 7.5 µL of 50 mM MgCl2 in DB was added to each vial and mixed. To prepare the assay plate, 15 µL of Vial A was dispensed into row A columns 1-4, 15 µL of Vial B was dispensed into row B columns 1-4 and 15 µL of Vial C was dispensed into row C columns 1-4. The Pherastar FSX was used to dispense 5 µL of 1.8 mM GTP in DB into each well. The plate was then shaken for 10 seconds and FI signal was measured every 30 seconds for 75 minutes. FI 485/520, Gain 300. FI data was analysed in Prism 7 (Version 7.04). The enzyme rate was calculated as the slope obtained by fitting a straight line in a range from 0-5 minutes, units are ΔRFU/time.
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