Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Neurotoxin B-IV
Abbreviated Name	B-IV
SCOP Family	Neurotoxin B-IV
Structure Notes
Organism	Cerebratulus lacteus (Milky ribbon worm)
UniProt Accession	P01525
SCOP Unique ID	n/a
Structure Solved
Class	Small
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	55
Molecular Weight	6107.2
Pi	9.5
Molecular Weight	6107.2
Disulphides	4
Full Sequence	ASATWGAAYPACENNCRKKYDLCIRCQGKWAGKRGKCAAHCIIQKNNCKGKCKKE
Notes	n/a

Expression
Report	Howell ML, Blumenthal KM. (1989) J Biol Chem, 264, 15268-73
Project Aim	Cloning and expression
Fusion	C-terminal T7 tag
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	2 h
Expression Vector	pSR9-X
Expression Protocol	Expression and Purification of Fusion Protein-Plasmid pSR9-X.B4 contains the toxin B-IV coding sequence fused to the 3\'-end of T7 gene 9 with a recognition sequence for protease Factor X. immediately upstream of B-IV (see \"Results\" for details of the construction). E. coli strain BL21(DE3) containing plasmid pSR9-XaB4 was grown at 37 \'C in Luria broth in the presence of 100 pg/ml ampicillin. Fusion protein synthesis was induced in late log phase (OD660 = 0.8-1) by addition of IPTG to 0.5 RIM. Cells were collected 2 h after induction by centrifugation, and the washed cell pellet was resuspended in 5 ml of 10 mM Tris-C1 (pH 8) containing 50 mM NaC1, 2 mM EDTA, and 5 mg of lysozyme per liter of culture. After 20 min on ice, 0-mercaptoethanol, phenylmethylsulfonyl fluoride, pepstatin, and leupeptin were added to final concentrations of 10 mM, 1 mM, 1 uM, and 0.5 uM, respectively. The cell suspension was frozen in dry ice/acetone and thawed awere performed at \"C.
Method of Induction	IPTG
Cell Density at Induction	OD 0.8-1.0 = 660
Cell Disruption Method	Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	DEAE-Cellulofine column chromatography
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	n/a
Solubilization Buffer	50 mM Tris-C1 (pH 7) containing 50 mM NaCl and 5 mM P-mercaptoethano
Refolding Buffer	0.1 M Tris-HC1 (pH 8.3) containing 200 mM NaCl and 1 mM EDTA, 1 mM oxidized, and 0.1 mM reduced glutathione
Pre-Refolding Purification	DEAE-Cellulofine column chromatography
Tag Cleaved	no
Refolding pH	8.3
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	overnight
Redox Agent	GSH/GSSG
Redox Agent Concentration	1/0.1 mM
Refolding Protocol	Reoxidation and Purification of B-IV-DEAE-purified fusion protein was dialyzed overnight at 4 \"C against two changes of 0.1 M Tris-HC1 (pH 8.3) containing 200 mM NaCl and 1 mM EDTA. After adjusting the protein concentration to 2 mg/ml, oxidized and reduced glutathione was added to final concentrations of 1 mM and 0.1 mM, respectively. Following overnight incubation at room temperature, the reoxidation mixture was dialyzed against 50 mM Tris-C1 (pH 8.3) containing 150 mM NaC1. Toxin B-IV was cleaved from the fusion protein by overnight hydrolysis at room temperature with protease Factor X. by the addition of phenylmethylsulfonyl fluoride to 1 mM. The (1 unit per 15 mg of fusion protein), and the reaction was terminated mixture was then dialyzed against 50 mM NH4HCOB and applied to a DEAE-cellulose column equilibrated in the same buffer. The flowthrough fractions, containing the basic B-IV protein were collected, lyophilized, and purified further by reverse-phase HPLC on a C4 column.
Refolding Assay	Biological assay
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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