Refolding Record:

Protein See all refolding records for this protein...
Protein Name	10 kDa chaperonin groES protein
Abbreviated Name	groES
SCOP Family	GroES
Structure Notes
Organism	Escherichia coli
UniProt Accession	P0A6F9
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	97
Molecular Weight	10387.0
Pi	5.14
Molecular Weight	10387.0
Disulphides	Unknown
Full Sequence	MNIRPLHDRVIVKRKEVETKSAGGIVLTGSAAAKSTRGEVLAVGNGRILENGEVKPLDVKVGDIVIFNDGYGVKSEKIDNEEVLIMSESDILAIVEA
Notes	n/a

Expression
Report	Okuda H, Sakuhana C, Yamamoto R, Kawai R, Mizukami Y, Matsuda K. (2007) Biometals, 20, 903-910
Project Aim	Folding
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	12 h
Expression Vector	pET22b
Expression Protocol	Recombinant GroEL was expressed in E. coli strain BL21 (DE3) (Novagen). The plasmid construct was used to transform E. coli BL21 (DE3), and the transformants were grown at 37°C in LB broth containing ampicillin (100 ug/ml). The protein expression was then induced by the addition of 0.5 mM IPTG. After incubation at 25°C for 12 h, the protein-overexpressing bacterial cells were harvested and resuspended in buffer A (50 mM Tris–HCl, pH 7.5, containing 1 mM EDTA and 50 mM NaCl). Lysozyme (0.5 mg/ml, Nacalai Tesque) and benzonase nuclease (5 units/ml, Novagen) were added to the cell suspension, which was subsequently disrupted by ultrasonication (Sonifire 450, Branson, CT, USA). The supernatant was fractionated by ammonium sulfate precipitation by a stepwise gradient from 30% (w/v) to 65% (w/v) saturation.
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	ultrasonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	gel filtration
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	50 mM Tris–HCl, pH 7.5, containing 1 mM EDTA and 50 mM NaCl
Solubilization Buffer	50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 250 mM NaCl, 20% (v/v) ethanol
Refolding Buffer	50 mM MOPS-KOH, pH 7.0, 10 mM KCl
Pre-Refolding Purification	gel filtration
Tag Cleaved	no tag
Refolding pH	7.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	The precipitates resulting from 55% ammonium sulfate saturation were harvested and dissolved in buffer A. The protein solution was then dialyzed overnight with 100-fold volume of buffer A with two buffer changes. The dialyzed proteins were loaded onto a HiPrep Q anion exchange column with buffer A and eluted by a linear gradient of NaCl from 50 mM to 1 M. Fractions containing GroES were pooled and concentrated by ultrafiltration using a 10-kDa cutoff filter (Millipore). Concentrated GroES was dialyzed overnight with 100-fold volumes of buffer C (50 mM acetic acid buffer, pH 5.3) with two buffer changes and the insoluble matter was removed from the dialyzed proteins by centrifugation. The resultant supernatants were loaded onto a Mono Q anion exchange column with buffer C and eluted with a linear gradient of NaCl from 0 mM to 400 mM. Moreover, the fraction containing GroES were purified using a Mono Q anion exchange column with 4-column volume of buffer A. The proteins were eluted with a linear gradient of NaCl from 50 mM to 1 M. Finally, GroES-containing fractions were purified by gel filtration using a Superdex 200 GL column (Amersham Biosciences) with buffer B. The fractions containing 7 meric GroES proteins were pooled and dialyzed with refolding buffer. The purity of the proteins was checked by SDS and native PAGE as described for GroEL.
Refolding Assay	SDS-PAGE
Refolding Chaperones	GroES
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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