Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Single chain Fv 125E11
Abbreviated Name	125E11
SCOP Family	V set domains (antibody variable domain-like)
Structure Notes
Organism	Mouse
UniProt Accession	Q66K04
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	246
Molecular Weight	26698.6
Pi	7.76
Molecular Weight	26698.6
Disulphides	Unknown
Full Sequence	QVQLQESGGGLVQPEGSLKLSCAASGFTFNTYAMHWVRQTPGKGLEWVARIRNKSNNYATYYADSVKDRFTISRDDSQNMLYLQMNNLKTEDTAMYYCVREASGMDYWGQGTTVTVS SGGGGSGGGGSGGGGS DIQLTQSPSSLAVSVGEKVSMSCKSSLNLLYNNNQLNYLAWYQQKPGQSPKLLISWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYPYTFGGGTKLEIK
Notes	UniProt reference is "best match" only- 84% identity to VH chain.

Expression
Report	Yang X, Hu W, Li F, Xia H, Zhang Z (2005) Protein Expression and Purification, 41, 341-348
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	BL21(DE3)
Expression Temp	37.0
Expression Time	3h
Expression Vector	pET32b+
Expression Protocol	200ml LB medium was inoculated with 3ml overnight stock and grown at 37degC. When OD600 reached 0.8-1.0, IPTG was added to 0.5mM and cells were grown for a further 3h. The cells were harvested by centrifugation (4000rpm, 10min, 4degC) and then about 1.6g of wet cells were resuspended in 25ml PBS.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.8-1.0
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Washing inclusion body
Solubility	insoluble

Refolding
Refolding Method	Gel filtration with pH and denaturant gradients
Wash Buffer	1: 0.5M NaCl, 1% Triton X-100, 2:20mM EDTA, 50mM Tris pH 8.0
Solubilization Buffer	8Murea, 1mM EDTA, 0.2M DTT, 20mM Tris pH 7.5
Refolding Buffer	0.2M urea, 1mM EDTA, 20mM Tris pH 7.5
Pre-Refolding Purification	Washing inclusion body
Tag Cleaved	no tag
Refolding pH	7.5
Refolding Temperature	25.0
Protein Concentration	5mg/ml
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Cells resuspended in PBS were sonicated and the lysate was centrifuged (16000rpm, 20min). The pellet was resuspended in wash buffer 1 and kept at room temperature for 30min. after centrifugation, the pellet was washed again in wash buffer 1, followed by two washes in wash buffer 2. The inclusion body pellet was denatured in and dissolved overnight at room temperature in solubilization buffer. The mixture was then centrifuged (16000rpm, 20min) and the supernatant was loaded onto a PD-10 column which was equilibrated with 8M urea, 1mM EDTA, 20mM Tris pH 2. Following this buffer exchange, the protein was passed down a Sephacryl S-100 column equilibrated in 0.2M urea, 1mM EDTA, 20mM Tris pH 7.5. A descending urea gradient and ascending pH gradient was created in the upper 30% of the column using two different solutions from 0.2M urea, 1mM EDTA, 20mM Tris pH 7.5 (refolding buffer) to 8M urea, 1mM EDTA, 20mM Tris pH 2. 1ml of the protein sample at 5mg/ml was applied to the column and eluted in refolding buffer.
Refolding Assay	Ligand Binding,Bradford assay
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	2.7mg/L culture
Purity	n/a
Notes	Refolding efficiency about 11%

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