mhhhhhhssgvdlgtenlyfqsmMQKLVVTRLSPNFREAVTLSRDCPVPLPGDGDLLVRNRFVGVNASDINYSAGRYDPSVKPPFDIGFEGIGEVVALGLSASARYTVGQAVAYMAPGSFAEYTVVPASIATPVPSVKPEYLTLLVSGTTAYISLKELGGLSEGKKVLVTAAAGGTGQFAMQLSKKAKCHVIGTCSSDEKSAFLKSLGCDRPINYKTEPVGTVLKQEYPEGVDVVYESVGGAMFDLAVDALATKGRLIVIGFISGYQTPTGLSPVKAGTLPAKLLKKSASVQGFFLNHYLSKYQAAMSHLLEMCVSGDLVCEVDLGDLSPEGRFTGLESIFRAVNYMYMGKNTGKIVVELPH
Procedure: The clear supernatant after centrifugation was passed through a Ni-NTA (2.5mL resin) column twice. The column was washed with 50 mL of wash buffer ( 500 mM NaCl, 5% Glycerol, 50 mM Tris-HCl pH 7.5, 30 mM Imidazole ), and protein was eluted with 15 mL of elution buffer ( 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 250mM Imidazole).
Column 2 : Hiload 16/60 Superdex 200 prep grade 120 mL, GE Healthcare.
Buffer : 10 mM HEPES, pH 7.5, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP.
Procedure: The eluted fractions from the Ni-affinity HisTrap columns were loaded on the gel filtration column in GF buffer at 1.0 mL/min. Eluted proteins were collected in 1 mL fractions
Concentration : 5 mg/mL using Vivaspin 10K concentrators
Procedure: The clear supernatant after centrifugation was passed through a Ni-NTA (2.5mL resin) column twice. The column was washed with 50 mL of wash buffer ( 500 mM NaCl, 5% Glycerol, 50 mM Tris-HCl pH 7.5, 30 mM Imidazole ), and protein was eluted with 15 mL of elution buffer ( 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 250mM Imidazole).
Column 2 : Hiload 16/60 Superdex 200 prep grade 120 mL, GE Healthcare.
Buffer : 10 mM HEPES, pH 7.5, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP.
Procedure: The eluted fractions from the Ni-affinity HisTrap columns were loaded on the gel filtration column in GF buffer at 1.0 mL/min. Eluted proteins were collected in 1 mL fractions
Concentration : 5 mg/mL using Vivaspin 10K concentrators
NMR Spectroscopy:
Data Collection:
Data Processing: