Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Mavicyanin
Abbreviated Name	n/a
SCOP Family	Plastocyanin/azurin-like
Structure Notes
Organism	Cucurbita pepo (Vegetable marrow) (Summer squash)
UniProt Accession	P80728
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	109
Molecular Weight	11747.1
Pi	7.94
Molecular Weight	11747.1
Disulphides	1
Full Sequence	ATVHKVGDSTGWTTLVPYDYAKWASSNKFHVGDSLLFNYNNKFHNVLQVDQEQFKSCNSSSPAASYTSGADSIPLKRPGTFYFLCGIPGHCQLGQKVEIKVDPGSSSA
Notes	n/a

Expression
Report	Kataoka K, Nakai M, Yamaguchi K, Suzuki S. (1998) Biochemical and Biophysical Research Com, 250, 409-413
Project Aim	Recombinant Protein Expression
Fusion	N-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	5 h
Expression Vector	pMAV1
Expression Protocol	Expression and purification of recombinant mavicyanin. Escherichia coli BL21(DE3) cells carrying pMAV1-1 were grown at 37 °C in 1 l of LB broth containing 50 mg/ml ampicillin until OD600 􏰂 0.5 􏰁 1.0, and induced with 1 mM IPTG. After another 5 h of incubation at the same temperature, the cells were harvested by centrifugation (3000 x g, 10 min) and washed twice with 30 mM Tris-HCl buffer (pH 7.5) containing 30 mM NaCl (buffer A). The cells (wet weight, 4 g) suspended in 20 ml of buffer A were disrupted well with KUBOTA insonater 201M ultrasonic oscillator. After centrifugation, the pellets (inclusion bodies) were washed with 1 M glucose (20 ml) and suspended in 2% Triton X-100, 10 mM EDTA solution (100 ml), being left overnight at 4 °C.
Method of Induction	IPTG
Cell Density at Induction	OD 0.5-1.0 = 600
Cell Disruption Method	Ultrasonic oscillator
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	1 M glucose (20 ml) and suspended in 2% Triton X-100, 10 mM EDTA solution (100 ml)
Solubilization Buffer	30 mM Tris-HCl pH 7.5, 30 mM NaCl, 1 mM DTT
Refolding Buffer	30 mM Tris-HCl pH 7.5, 30 mM NaCl, 1 mM DTT, 4 M urea
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	7.5
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	12 h
Redox Agent	DTT
Redox Agent Concentration	1 mM
Refolding Protocol	The washed pellets were solubilized with 5 ml of buffer B (30 mM Tris-HCl pH 7.5, 30 mM NaCl, 1 mM DTT) supplemented 8 M urea, and dialyzed against buffer B containing 4 M urea at 4 °C for 12 h. After another 24 h dialysis against buffer B, solubilized crude recombinant apoprotein was econstituted by dialysis against 20 mM Tris-HCl (pH 7.5) containing 1 mM CuSO4 for overnight at 4 °C. Reconstituted protein was dialyzed against 10 mM potassium phosphate buffer (pH 7.0) and applied onto a CM-Sephadex column (􏰆2.5 x 12.5 cm) preequilibrated with the same buffer. After the unabsorbed proteins were washed out with the 10 mM buffer, the recombinant mavicyanin was eluted with 20 mM buffer. The fractions containing mavicyanin identified by SDS-PAGE analysis were pooled and the recombinant protein thus purified was used for subsequent experiments.
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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