PDB:4C8D
Revision
Revision Type: created
Revised by: created
Revision Date: created
Entry Clone Accession: NM_016604
Entry Clone Source:
SGC Clone Accession:
Tag: C-terminal, TEV cleavable hexahistidine tag
Host:
Sequence: MTSHSWLCDGRLLCLHDPSNKNNWKIFRECWKQGQPVLVSGVHKKLKSELWKPEAFSQEFGDQDVDLVNCRNCAIISDVKVRDFWDGFEIICKRLRSEDGQPMVLKLKDWPPGEDFRDMMPTRFEDLMENLPLPEYTKRDGRLNLASRLPSYFVRPDLGPKMYNAYGLITAEDRRVGTTNLHLDVSDAVNVMVYVGIPIGEGAHDEEVLKTIDEGDADEVTKQRIHDGKEKPGALWHIYAAKDAEKIRELLRKVGEEQGQENPPDHDPIHDQSWYLDQTLRKRLYEEYGVQGWAIVQFLGDAVFIPAGAPHQVHNLYSCIKVAEDFVSPEHVKHCFRLTQEFAGNLYFQ
Note: a change from E to G within the TEV protease site does not affect cleavage.
Vector:pNIC-CTHF
Medium:
Antibiotics:
Procedure:
Buffers
Procedure
Lysis/ Binding Buffer: 50mM HEPES pH 7.4, 500mM NaCl, 5% glycerol, 10 mM Imidazole pH 7, 0.5 mM TCEP, mixture of proteinase inhibitors.
Wash Buffer : 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 40 mM Imidazole pH 7.4, 0.5mM TCEP
Elution Buffer:50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.4, 0.5mM TCEP
Gel Filtration buffer: 20 mM Hepes pH 7.4, 500 mM NaC, 5% glycerol, 0.5mM TCEP
Cell Lysis: The cells from one litre culture, were lysed by sonication. Cell debris and DNA were spun down at 56000x g, 45 min ( Beckman JA 50.50 21500 rpm). The supernatant was collected to which Benzonase was added ( 1ul/litre culture).
Affinity binding to Ni-sepharose resin: The clarified cell extract was first batch bound to 2ml resin /litre culture. The slurry was rotated for 90 minutes at 4°C, then spun 15min/500xg/ 4°C.The supernatant was removed. The pelleted resin was loaded onto a glass column. The column was washed with 30CV of binding buffer, 5CV of wash buffer, and then eluted in aliquots with elution buffer.
TEV digestion: TEV was added to the eluted protein at a molar ratio of 1:20. The digestion was continued over night at 4C. The complete digestion was confirmed by mas spec.
Gel filtration, Hiload 16/60 Superdex S200 prep grade, 120 ml The digested protein was loaded on a gel filtration column, pre-equilibrated in GF buffer, at 1.2 ml/min. Eluted proteins were collected in 1.75 ml fractions and analyzed on SDS-PAGE.
Rebinding of the gel filtration pool: The pool was loaded onto a column with 0.4 ml Ni-sepharose, collecting the flow through and a gel filtration buffer wash.
Concentrating: The flow through was concentrated using a centricon centrifugal device with a 30kDa MWCO.The concentrated protein was centrifuged 10 min/ 14000rpm/4C. The final concentration, in the supernatant, was 24.1 mg/ml and yield 20 mg/litre culture. The protein was flash frozen and stored at -80°C in 70ml aliquots.
Buffers
Procedure
Expression strain: BL-21(DE3)-R3-Rosetta (A homemade phage resistant version of BL21(DE3) containing the pRARE2 plasmid from Rosetta II (DE3) cells).
Transformation: The construct DNA was transformed into competent cells of the expression strain by a standard heat shock procedure.
Glycerol stock preparation: A number of colonies were used to inoculate 1 ml of LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was incubated in a 37°C shaker overnight. The next day glycerol stocks were prepared from this overnight culture.
Expression: 10 ml of a glycerol stock was used to inoculate 120 ml of LB containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was incubated at 37°C overnight. 10ml starter culture was used per litre TB, containing 50 µg/ml kanamycin. The culture was incubated at 37°C until OD~1.5, when the temperature of the incubator was reduced to 18°C. At 18°C, expression was induced with 0.1 mM IPTG and the culture continued o/n.
Cell harvest: Cells were pelleted at 6238x g for 15 min at 4°C, resuspended in lysis buffer (50mM HEPES pH7.4,10mM imidazole, 0.5M NaCl, 5% glycerol, 0.5mM TCEP and proteinase inhibitors), flash frozen in liquid nitrogen and stored at -80°C.
Concentration:
Ligand
MassSpec:Mass spec. characterisation: Measured: 40092.75 and 40224.10 Expected: 40294.8
Crystallization:0.50mM JMJD1BA-p056 was incubated with 2mM NOG and 6mM MnCl2 for 2.5 hrs, on ice. The protein was spun 10min/21000xg and the supernatant was used for setting up crystallization plates. The crystal was found in precipitant [0.1M BisTris buffer pH=5.5, 0.1M ammonium acetate, 27% PEG3350]. The cryo contained 25% glycerol and 75% precipitant.
NMR Spectroscopy:
Data Collection:Data was collected to a resolution of 2.5 Å, at Diamond beamline.
Data Processing: