Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Cholera enterotoxin subunit A [Precursor]
Abbreviated Name	CTA1
SCOP Family	Bacterial AB5 toxins, B-subunits
Structure Notes
Organism	Vibrio cholerae
UniProt Accession	P01555
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Unknown

Construct
Full Length	n
Domain	Subunit A1
Chimera	n/a
Variants	n/a
Chain Length	194
Molecular Weight	21815.0
Pi	6.03
Molecular Weight	21815.0
Disulphides	1
Full Sequence	NDDKLYRADSRPPDEIKQSGGLMPRGQSEYFDRGTQMNINLYDHARGTQTGFVRHDDGYVSTSISLRSAHLVGQTILSGHSTYYIYVIATAPNMFNVNDVLGAYSPHPDEQEVSALGGIPYSQIYGWYRVHFGVLDEQLHRNRGYRDRYYSNLDIAPAADGYGLAGFPPEHRAWREEPWIHHAPPGCGNAPRSS
Notes	n/a

Expression
Report	Ampapathi RS, Creath AL, Lou DI, Craft JW Jr, Blanke SR, Legge GB. (2008) Journal of Molecular Biology, 1, 1
Project Aim	Undefined
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	4 h
Expression Vector	n/a
Expression Protocol	CTA1 protein expression and purification CTA1 and CTA1-T2 were overexpressed in E. coli BL21(DE3) transformed with plasmids harboring the genes encoding these proteins. For unlabeled samples, E. coli BL21(DE3) was cultured in LB supplemented with 50 μg/mL of ampicillin. 15N-labeled and 15N/13C-labeled protein samples were generated by culturing E. coli BL21(DE3) in M9 minimal media supplemented with 50 μg/mL of ampicillin and containing 15NH4Cl as well as 15(NH4)2SO4 and 13C-labeled glucose (Sigma-Aldrich) as the sole nitrogen and carbon sources, respectively. 2H/15N/13C-labeled CTA1-T2 was generated by first culturing transformed E. coli BL21(DE3) in 10 mL of M9 minimal media supplemented with 50 μg/mL of ampicillin and containing 15NH4Cl, 15(NH4)2SO4, and 13C-labeled glucose. After incubation for 4 h at 37 °C, the cells were harvested by centrifugation at 4000 rpm in an F21B-8x50Y rotor (FIBERLite, Piramoon Technologies, Inc.); the available pellet was resuspended in M9 in 2H2O and added to 1 L of M9 in 2H2O with 50 μg/mL of ampicillin. Cells were cultured to mid log phase (with OD600 being approximately 0.6), expression was induced with 0.1 mM IPTG, and expression continued overnight at 37 °C.
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	50 mM Tris, pH 8.0, 2 mM EDTA, 2 mM DTT, 200 μg/mL of lysozyme (Sigma-Aldrich), and 10% B-PER
Solubilization Buffer	6 M Gdm–HCl and 50 mM Tris, pH 8.0,
Refolding Buffer	20 mM Tris, pH 7.2, 100 mM NaCl, and 5% glycerol
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	7.2
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a,n/a
Refolding Protocol	The resuspended cells were sonicated on ice and centrifuged at 13,000 rpm; the pellets were washed several times with homogenization buffer with a final wash in ultrapure water. The inclusion bodies were solubilized by suspending the pellets in 6 M Gdm–HCl and 50 mM Tris, pH 8.0, and allowed to incubate overnight at 4 °C with mixing. The solubilized proteins were renatured by dialysis against refolding buffer (20 mM Tris, pH 7.2, 100 mM NaCl, and 5% glycerol) at 4 °C. The refolded protein samples were centrifuged as previously described, filtered (0.45 μm), and purified at 4 °C using the ÄKTA™ FPLC System with a HiLoad™ 16/60 Superdex™ 75 prep grade gel filtration column (GE Healthcare).
Refolding Assay	Unspecified,Gel filtration chromatography
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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