Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Botulinum neurotoxin type A
Abbreviated Name	BoNT-A
SCOP Family	Clostridium neurotoxins, C-terminal domain
Structure Notes
Organism	Clostridium botulinum
UniProt Accession	Q7B8V4
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Monomer

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	247
Molecular Weight	27882.5
Pi	9.23829
Molecular Weight	27882.5
Disulphides	0
Full Sequence	MGSSHHHHHHSSGLVPRGSHM MSNSGILKDF WGDYLQYDKP YYMLNLYDPN KYVDVNNVGI RGYMYLKGPR GSVMTTNIYL NSSLYRGTKF IIKKYASGNK DNIVRNNDRV YINVVVKNKE YRLATNASQA GVEKILSALE IPDVGNLSQV VVMKSKNDQG ITNKCKMNLQ DNNGNDIGFI GFHQFNNIAK LVASNWYNRQ IERSSRTLGC SWEFIPVDDG WGERPL DPAANKARKEAELAAATAEQ
Notes	n/a

Expression
Report	Sharma S, Zhou Y, Singh BR (2006) Protein Expression and Purification, 45, 288-95
Project Aim	Recombinant Protein 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(DE3)Star
Expression Temp	30.0
Expression Time	15h
Expression Vector	pET15b
Expression Protocol	Cells were grown at 37degC until OD600 reached 0.8. IPTG was added to 1.0mM, and cells were incubated at 30degC for a further 15h. The cells were then harvested by centrifugation (8000g, 10min, 4degC) and the cell pellet was stored at -80degC.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.8
Cell Disruption Method	Chemical
Lytic Agent	Lysozyme
Pre-Refolding Purification	Washing inclusion body
Solubility	insoluble

Refolding
Refolding Method	Column refolding: Nickel-chelating chromatography
Wash Buffer	1mM TrisHCl, mild nonionic detergent - 1:20 dilution of CelLytic B II (Sigma)
Solubilization Buffer	0.1M sodium phosphate pH 8.0, 6M GdnHCl, 0.4M NaCl, 10mM 2-mercaptoethanol
Refolding Buffer	0.1M sodium phosphate pH 8.0, 0.4M NaCl, 0.1% dodecylphosphocholine
Pre-Refolding Purification	Washing inclusion body
Tag Cleaved	no
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	Beta-mercaptoethanol
Redox Agent Concentration	10mM,10mM
Refolding Protocol	The cell pellet was thawed and resuspended in lysis buffer - CelLytic B II (20mM TrisHCl pH 7.5 and a mild nonionic detergent) at a ration of 5ml buffer per gram of wet cell paste. After resuspension of the cell pellet, 5microg/ml DNaseI and a protease inhibitor cocktail were added to the suspension at 1ml for 20g wet cell paste. The suspension was incubated at 25degC for 15min and centrifuged (25000g, 15min). The pellet was resuspended again in lysis buffer and lysozyme was added to a concentration of 0.4mg/ml. The mixture was incubated at 25degC for 15min and 20ml of 1:20 diluted lysis buffer (wash buffer) was added. The suspension was incubated a further 15min at 25degC and centrifuged(25000g, 15min). The inclusion body pellet was then resuspended in 40ml wash buffer and centrifuged again (25000, 15min), a process which was repeated two more times. The inclusion bodies were resuspended in 25ml solubilization buffer. Protease inhibitor cocktail was added at 1ml for 20g wet weight cell paste and the suspension was kept on a rocker for 1h at 25degC. The solution was then centrifuged (1h, 100000g, 4degC) and the supernatant was diluted 1:1 with equilibration buffer (8M urea, 0.4M NaCl, 5mM imidazole, 10mM 2-mercaptoethanol, 0.1M sodium phosphate pH 8.0). The protein was then purified under denaturing conditions using a His-Select nickel affinity column. The protein was step-eluted in refolding buffer containing increasing concentrations of imidazole up to 100mM
Refolding Assay	Ligand Binding,Western Blot,SDS-PAGE
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	11mg/L culture
Purity	95%
Notes	n/a

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