retSDR3
PDB:1YDE
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi:7705907
Entry Clone Source:Synthetic
SGC Clone Accession:Tag:N-term: gsshhhhhhssgrenlyfqghm. C-term: gs
Host:Rosetta-2 (DE3)
Construct
Prelude:Sequence:ATGTRYAGKVVVVTGGGRGIGAGIVRAFVNSGARVVICDKDESGGRALEQELPGAVFILCDVTQEDDVKTLVSETIRRFGRLDCVVNNAGHHPPPQRPEETSAQGFRQLLELNLLGTYTLTKLALPYLRKSQGNVINISSLVGAIGQAQAVPYVATKGAVTAMTKALALDESPYGVRVNCISPGNIWTPLWEELAALMPDPRASIREGMLAQPLGRMGQPAEVGAAAVFLASEANFCTGIELLVTGGAELGYGCKASRSTPVDAPDIPS
Vector:p11
Growth
Medium:Antibiotics:Procedure:Medium: TB, 34 µg/mL kanamycin and 100 µg/mL ampicillin. 1 liter TB in 2.5 L baffled flasks was inoculated with an overnight culture. The culture was grown at 37 °C to OD of 0.6 and then transfered to 15 °C. 1 mM IPTG was then added, and incubation continued over a period of 20 hours. The cells were then collected by centrifugation and frozen at -80°C
Purification
ProcedureBuffers for DE-52 and Ni-NTA columns: Binding buffer (BB): 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM imidazole. Wash buffer (WB): 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% glycerol, 30 mM imidazole. Elution buffer (EB): 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 250 mM imidazole. Procedure: Gravity feed chromatography. Sample applied to a 10 ml DE-52 column and washed through with 20 mL BB. The flow through was applied to a 2 mL Ni-NTA column, the Ni-NTA column was washed with 200 mL of WB and eluted with EB in 2 mL aliquots. Eluate was monitored for protein using Coomassie Blue Plus protein assay reagent. Procedure: As above.
Gel Filtration: S75 16/60 prep grade.
GF buffer: 10 mM HEPES, pH 7.5 500 mM NaCl, 5% glycerol, 0.5 mM TCEP. Procedure: The eluted fraction was loaded and fractionated on the gel filtration column in GF buffer at 1 mL/min.
Extraction
ProcedureBuffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 5 mM Imidazole, PMSF to 1 mM added.
Concentration:LigandMassSpec:Crystallization:Magnesium acetate, MPD, 0.1M cacodylate buffer, pH 6.5, vapour diffusion, hanging drop, temperature 293K
NMR Spectroscopy:Data Collection:Data Processing:
