Refolding Record:

Protein See all refolding records for this protein...
Protein Name	TNF-related apoptosis-inducing ligand
Abbreviated Name	TRAIL
SCOP Family	TNF-like
Structure Notes
Organism	Human
UniProt Accession	P50591
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Monomer

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	281
Molecular Weight	32508.9
Pi	7.00603
Molecular Weight	32508.9
Disulphides	0
Full Sequence	MAMMEVQGGP SLGQTCVLIV IFTVLLQSLC VAVTYVYFTN ELKQMQDKYS KSGIACFLKE DDSYWDPNDE ESMNSPCWQV KWQLRQLVRK MILRTSEETI STVQEKQQNI SPLVRERGPQ RVAAHITGTR GRSNTLSSPN SKNEKALGRK INSWESSRSG HSFLSNLHLR NGELVIHEKG FYYIYSQTYF RFQEEIKENT KNDKQMVQYI YKYTSYPDPI LLMKSARNSC WSKDAEYGLY SIYQGGIFEL KENDRIFVSV TNEHLIDMDH EASFFGAFLV G
Notes	No fusion tag stated, but a Ni-NTA purification step performed

Expression
Report	Shen YL, Xia XX, Zhang Y, Liu JW, Wei DZ, Yang SL (2003) biotechnology letters, 25, 2097-2101
Project Aim	Recombinant Protein Expression
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	C600
Expression Temp	42.0
Expression Time	4h
Expression Vector	pBV
Expression Protocol	Cells were grown at 30degC in a fermenter, the pH was controlled at 7 and dissolved O2 maintained above 25%. When the culture reached 65g dry cells per litre, the temperature was shifted to 42degC and incubation continued for 4h. The cells were harvested by centrifugation (9000g, 4degC, 30min)
Method of Induction	Temperature Shift
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	High pressure homogenization
Lytic Agent	None
Pre-Refolding Purification	Washing inclusion body
Solubility	insoluble

Refolding
Refolding Method	Dilution/Dialysis combination
Wash Buffer	0.05M sodium phosphate pH 7.4, 1M NaCl, 6M urea, 0.5% *(v/v) Triton X-100
Solubilization Buffer	0.05M TrisHCl pH 8.5, 5M GdnHCl, 0.03M DTT
Refolding Buffer	1:0.05M TrisHCl pH 7.4, 0.4M L-arginine, 2mM DTT, 0.5M urea, 0.5M NaCl 2: 0.05M TrisHCl pH 7.4, 0.3M NaCl, 1mM DTT, 0.1M urea
Pre-Refolding Purification	Washing inclusion body
Tag Cleaved	no tag
Refolding pH	7.4
Refolding Temperature	4.0
Protein Concentration	up to 1mg/ml
Refolding Time	o/night
Redox Agent	DTT
Redox Agent Concentration	1-2mM,1-2mM
Refolding Protocol	Cell pellets were suspended at 4degC in 0.05M sodium phosphate pH 7.4, 0.02M EDTA, 3% Triton X-100. Cells were lysed by high-pressure homogenization and inclusion bodies were collected by centrifugation (15000g, 30min). The inclusion bodies pellet was washed with wash buffer then stored at -70degC. The pellet was then resuspended in solubilization buffer and stirred at 4degC for 2h. Insoluble material was removed by centrifugation (25000g, 20min) and the supernatant was diluted with solubilization buffer to a final protein concentration of 7.2mg/ml. The protein was refolded by step-wise dilution in 200ml refolding buffer. At each step, 30ml unfolded protein was added to refolding buffer 1 at about 180microg/ml per step, with an interval of 60min per addition. The solution was then dialyzed overnight against refolding buffer 2 at 4degC. The solution was centrifuged (25000g, 30min, 4degC) and the supernatant retained for further purification
Refolding Assay	Bioactivity,SDS-PAGE
Refolding Chaperones	None
Refolding Additives	L-Arginine
Additives Concentration	0.4M
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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