Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Streptavidin
Abbreviated Name	SCD
SCOP Family	Avidin/streptavidin
Structure Notes
Organism	Streptomyces avidinii
UniProt Accession	P22629
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Tetramer

Construct
Full Length	n
Domain	core protein, aa.13-139
Chimera	n/a
Variants	Circular permutation from aa.51(N) to 46(C)
Chain Length	139
Molecular Weight	14402.6
Pi	6.1
Molecular Weight	14402.6
Disulphides	0
Full Sequence	ESRYVL TGRYDSAPAT DGSGTALGWT VAWKNNYRNA HSATTWSGQY VGGAEARINT QWLLTSGTTE ANAWKSTLVG HDTFTKVKPS AAS GGGS DPSKDS KAQVSA AEAG ITGTWYNQLG STFIVTAGAD GALTGTYESA
Notes	n/a

Expression
Report	Chu V, Freitag S, Le Trong I, Stenkamp RE, Stayton PS (1998) Protein Science, 7, 848-859
Project Aim	Crystallography,Structural Studies
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	pET-21a
Expression Protocol	6.5L 2xTY broth with 100microg/ml ampicillin was inoculated with 10ml overnight culture. Cells were grown at 37degC until OD600 reached 1.0, then 1mM IPTG was added. Cells were cultured for a further 3h, then harvested by centrifugation (4500g, 10min)
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 1.0
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Washing inclusion body
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	50mM TrisHCl pH 8.0, 5mM EDTA, 200mM NaCl, 1mM PMSF, 8% sucrose, 0.5% Triton X-100
Solubilization Buffer	6M GdnHCl, 50mM TrisHCl pH 7.5
Refolding Buffer	50mM TrisHCl pH 7.5, 100mM NaCl, 5mM EDTA, 0.1mM PMSF
Pre-Refolding Purification	Washing inclusion body
Tag Cleaved	no tag
Refolding pH	7.5
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	overnig
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	The cell pellet was resuspended in wash buffer, sonicated and centrifuged (17700g, 20min). The insoluble fraction was sonicated and centrifuged twice more in wash buffer, then three times in wash buffer without Triton X-100. The protein was then dssolved in solubilization buffer to a concentration of no more than 10mg/ml and allowed to equilibrate for several hours at 4degC. The protein was then diluted dropwise with stirring at 4degC in a 50x volume of refolding buffer and was allowed to equilibrate overnight. The resulting solution was centrifuged to remove insoluble matieral, then concentrated and purified further by affinity chromatography using iminobiotin-agarose.
Refolding Assay	Crystallography,Amino acid sequencing,Mass spectrometry,Isothermal titration calorimetry
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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