Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Cholera enterotoxin subunit B Also known as Cholera toxin B subunit
Abbreviated Name	CTB
SCOP Family	Bacterial AB5 toxins, B-subunits
Structure Notes
Organism	Vibrio cholerae
UniProt Accession	P01556
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Pentamer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	104
Molecular Weight	11645.4
Pi	7.72
Molecular Weight	11645.4
Disulphides	Unknown
Full Sequence	TPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN
Notes	n/a

Expression
Report	Arêas AP, Oliveira ML, Ramos CR, Sbrogio-Almeida ME, Raw I, Ho PL. (2002) Protein Expression and Purification, 25, 481-487
Project Aim	Cloning and expression
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(SI)
Expression Temp	30.0
Expression Time	3-16 h
Expression Vector	pAE
Expression Protocol	BL21 (SI) E. coli competent cells (Gibco/BRL) were transformed with the pAE–ctxB plasmid and grown overnight at 30 °C. In this strain, the expression of T7 RNA polymerase is under the control of the E. coli osmotically inducible promoter proU [28]. Therefore, the production of recombinant proteins is induced by the addition of NaCl to the medium. Some ampicilin-resistant colonies were inoculated in 5 ml of LBON-amp (Luria–Bertani broth without NaCl) and grown overnight at 30 °C. Bacteria BL21 (SI) carrying the plasmid from an overnight culture were diluted 20-fold in LBON-amp broth. When A600 reached 0.6, NaCl was added to the medium at final concentrations of 100, 200, or 300 mM. After 3–16 h, cells were harvested by centrifugation and bacterial pellets were lysed by the addition of 2-fold SDS–PAGE sample buffer. Aliquots of total cellular extracts were analyzed by 15% SDS–PAGE. The best condition was chosen for expression in larger volume (1 L). The recombinant CTB was expressed in an insoluble form as inclusion bodies, as previously described [2].
Method of Induction	NaCl
Cell Density at Induction	OD 0.6 = 600
Cell Disruption Method	French Press
Lytic Agent	Detergents
Pre-Refolding Purification	not specified
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	(50 mM Tris–Cl and 500 mM NaCl) containing 10 mM β-mercaptoethanol and 2 M urea
Solubilization Buffer	(50 mM Tris–Cl and 500 mM NaCl) containing 10 mM β-mercaptoethanol , 8 M urea
Refolding Buffer	50 mM Tris–Cl and 500 mM NaCl, 5 mM imidazole
Pre-Refolding Purification	not specified
Tag Cleaved	no
Refolding pH	0.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	24 h
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	nclusion bodies were washed 2-fold with 15 ml of binding buffer (50 mM Tris–Cl and 500 mM NaCl) containing 10 mM β-mercaptoethanol and 2 M urea. Then, the inclusion bodies were dissolved in 10 ml binding buffer containing 10 mM β-mercaptoethanol and 8 M urea. The solubilized pellet was slowly diluted in 2 L of binding buffer containing 5 mM imidazole for CTB refolding and incubated for 24 h at room temperature.
Refolding Assay	Western Blot
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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