Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Tcell alpha chain
Abbreviated Name	TCR
SCOP Family	Unknown
Structure Notes
Organism	Human
UniProt Accession	A2NZL4
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Monomer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	101
Molecular Weight	11433.0
Pi	8.61
Molecular Weight	11433.0
Disulphides	Unknown
Full Sequence	MDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVVRDSSYKLIFGSGTRLLVRP
Notes	n/a

Expression
Report	Hilyard KL, Reyburn H, Chung S, Bell JI, Strominger JL. (1994) Proc. Natl. Acad. Sci. USA, 91, 9057-9061
Project Aim	Undefined
Fusion	C-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	W3110
Expression Temp	30.0
Expression Time	10-15 h
Expression Vector	pelBscHIS
Expression Protocol	To induce expression of TCR scFv, E. coli strain W3110 transformed with pelBscHIS was grown in Luria broth containing ampicillin (100 Mg/ml) and 1% (wt/vol) glucose at 30°C to a cell density at A550 of 0.5-1.0. Cells were then pelleted by centrifugation and washed once with Luria broth. The cells were resuspended in fresh Luria broth containing ampicillin (100 ,ug/ml) and 1 mM isopropyl ,B-D-thiogalactopyranoside, grown for a further 10-15 hr at 30°C, and then pelleted by centrifugation.
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Freeze-thaw
Lytic Agent	Lysozyme
Pre-Refolding Purification	Ni-NTA column
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	0.5% Triton X-100/50 mM Tris-HCl, pH 8.0/100 mM NaCl/0.1% (wt/vol) sodium azide.
Solubilization Buffer	8 M urea/100 mM NaH2PO4/10 mM Tris-HCl (urea buffer) at pH 8.0.
Refolding Buffer	3 M urea/50 mM Tris-HCl, pH 8.0/1 mM EDTA/3 mM oxidized glutathione/0.3 mM reduced glutathione/0.1 mM phenylmethylsulfonyl fluoridechromatography
Pre-Refolding Purification	Ni-NTA column
Tag Cleaved	no
Refolding pH	8.0
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	48 h
Redox Agent	GSH/GSSG
Redox Agent Concentration	0.3/3 mM,0.3/3 mM
Refolding Protocol	The supernatant was removed, and the cell pellet containing inclusion bodies and cell membrane was repeatedly washed in 0.5% Triton X-100/50 mM Tris-HCl, pH 8.0/100 mM NaCl/0.1% (wt/vol) sodium azide. After a final wash in 50 mM Tris HCl, pH 8.0/100 mM NaCl, the inclusion bodies were solubilized in 8 M urea/100 mM NaH2PO4/10 mM Tris-HCl (urea buffer) at pH 8.0. After centrifugation at 10,000 x g for 20 min to remove insoluble debris, the supernatant was loaded onto a nickel-nitrilotriacetic acid (Ni-NTA) column (Qiagen, Chatsworth, CA) and washed with 10 volumes of urea buffer at pH 8.0 and then 5 volumes of urea buffer at pH 6.5. Protein was eluted from the column in 5 volumes of urea buffer at pH 4.5. Fractions containing protein (as determined by A280) were pooled and diluted into the refolding buffer to a protein concentration of A280 = 0.03. The final refolding buffer was 3 M urea/50 mM Tris-HCl, pH 8.0/1 mM EDTA/3 mM oxidized glutathione/0.3 mM reduced glutathione/0.1 mM phenylmethylsulfonyl fluoride. The refolding solution was incubated at 4°C with gentle mixing for 48 hr and then dialyzed against 50 mM Tris HCl, pH 8.0/10 mM NaCl at 4°C for 24 hr. Refolded protein was concentrated and then passed down a desalting column (PD-10; Pharmacia) equilibrated in 10 mM Na2H2PO4, pH 8.0/10 mM NaCl to remove final traces of refolding buffer.
Refolding Assay	Far-UV Circular Dichroism,HPLC
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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