GLRX2
PDB:2FLS
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC009669
Entry Clone Source:Invitrogen LLAM 3879347
SGC Clone Accession:Tag:Tag sequence: mhhhhhhssgvdlgtenlyfqs*(m), TEV-cleavable (*), N-terminal his6 tag.
Host:Rosetta-R3
Construct
Prelude:Sequence:mhhhhhhssgvdlgtenlyfqsmPVNQIQETISDNCVVIFSKTSCSYCTMAKKLFHDMNVNYKVVELDLLEYGNQFQDALYKMTGERTVPRIFVNGTFIGGATDTHRLHKEGKLLPLVHQCYLKKSKRKEFQ
Vector:pNIC28-BSA4
Growth
Medium:Antibiotics:Procedure:Medium: TB + 50 µg/ml Kanamycin + 34 ug/ml chloramp . 2 x 1 liter TB in 2.5-L baffled flasks were inoculated with 10 ml overnight culture and grown grown at 37°C. The protein expression was induced with 1 mM IPTG at OD600 = 4.5 at 18°C overnight . The cells were collected by centrifugation and frozen at -80°C.
Purification
ProcedureColumn 1 : Ni-affinity, HisTrap, 1 ml (GE/Amersham Biosciences )
Buffers: Lysis buffer: 50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 10 mM imidazole; Wash buffer: 50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 50 mM imidazole; Elution buffer: 50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 250 mM imidazole.
Procedure: The cell extract was loaded on the column at 0.8 ml/minute on an AKTA-express system (GE/Amersham). The column was then washed with 10 volumes of lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 0.8 ml/min. The eluted peak of A280nm was automatically collected.
Column 2: Hiload 16/60 Superdex 75 prep grade 120 ml (GE/Amersham Biosciences)
Extraction
ProcedureLysis buffer: 50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 10 mM imidazole, Complete® protease inhibitors (1 tablet/50 ml). Frozen cell pellets were thawed on ice overnight and resuspended in a total volume of 100 ml lysis buffer. The cells were disrupted by high pressure (20 kpsi) followed by sonication. Nucleic acids and cell debris were removed by adding 0.15% PEI , followed by centrifugation for 30 minutes at 40 000xg. The supernatant was further clarified by filtration (0.20 m m).
Concentration:LigandMassSpec:Crystallization:Column 1 : Ni-affinity, HisTrap, 1 ml (GE/Amersham Biosciences )
Buffers: Lysis buffer: 50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 10 mM imidazole; Wash buffer: 50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 50 mM imidazole; Elution buffer: 50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 250 mM imidazole.
Procedure: The cell extract was loaded on the column at 0.8 ml/minute on an AKTA-express system (GE/Amersham). The column was then washed with 10 volumes of lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 0.8 ml/min. The eluted peak of A280nm was automatically collected.
Column 2: Hiload 16/60 Superdex 75 prep grade 120 ml (GE/Amersham Biosciences)
NMR Spectroscopy:
Data Collection:
Data Processing:
