Refolding Record:

Protein See all refolding records for this protein...
Protein Name	B lymphocyte stimulator
Abbreviated Name	BLyS/BAFF
SCOP Family	TNF-like
Structure Notes
Organism	Human
UniProt Accession	Q9Y275
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Monomer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	152
Molecular Weight	17040.5
Pi	4.92
Molecular Weight	17040.5
Disulphides	1
Full Sequence	AVQGPEE TVTQDCLQLI ADSETPTIQK GSYTFVPWLL SFKRGSALEE KENKILVKET GYFFIYGQVL YTDKTYAMGH LIQRKKVHVF GDELSLVTLF RCIQNMPETL PNNSCYSAGIAKLEEGDELQ LAIPRENAQI SLDGDVTFFG ALKLL
Notes	n/a

Expression
Report	Cao P, Mei JJ, Diao ZY, Zhang SQ (2005) Protein Expression and Purification, 41, 199-206
Project Aim	Functional Studies,Recombinant Protein Expression
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)
Expression Temp	37.0
Expression Time	5h
Expression Vector	pET30a(+)
Expression Protocol	Cells were grown in LB medium with shaking (220rpm) at 37degC to a density of OD600=0.6. 1mM IPTG was added and cells were grown a further 5h bfore being harvested by centrifugation (6000g, 15min, 4degC)
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.6
Cell Disruption Method	Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Column refolding: Size-exclusion chromatography
Wash Buffer	50mM TrisHCl, 5mM EDTA, 0.15M NaCl pH 8.0
Solubilization Buffer	50mM TrisHCl, 8M urea, 0.1M DTT pH 8.0
Refolding Buffer	50mM TrisHCl, 5mM EDTA, 150mM NaCl, 1.25mM GSH, 0.25mM GSSG pH 8.0
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	GSH/GSSG
Redox Agent Concentration	1.25mM/0.25mM,1.25mM/0.25mM
Refolding Protocol	2g wet weight of cell pellet was resuspended in 20ml of 50mM TrisHCl, 5mM EDTA, 0.15mM NaCl, 1mg/ml lysozyme pH 8.0, for 45min at 4degC. Cells were then lysed by sonication in the presence of 15mM PMSF. Inclusion bodies were collected by centrifugation (10000g, 10min, 4degC). The inclusion body pellet was washed firstly in 40ml wash buffer, centrifuged, then in 40ml of wash buffer with 3M urea and centrifuged again. This 2nd wash step was repeated three more times. The inclusion bodies were washed in 40ml of wash buffer containing 0.5% Triton X-100(v/v) and centrifuged (13000g, 15min) four times. The inclusion bodies were then dissolved in 8ml of solubilization buffer for 12h at room temperature. The protein was then refolded by passage down a Sephacryl S-200 column. The column was equilibrated with wash buffer, then filtered unfolded protein was passed through the column in refolding buffer at a rate of 0.5ml/min. Selected fractions were then dialyzed against 50mM TrisHCl, 50mM NaCl pH 7.2 for 24h at 4degC. The protein was then further purified using a DEAE sepharose column at 4degC.
Refolding Assay	Far-UV Circular Dichroism,Fluorescence,Bioactivity,Western Blot,SDS-PAGE,Amino acid sequencing,Mass spectrometry
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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