Refold - Enhanced Green Fluorescent Protein

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Enhanced Green Fluorescent Protein
Abbreviated Name	EGFP
SCOP Family	Fluorescent proteins
Structure Notes
Organism	Aqueorea victoria
UniProt Accession	P42212
SCOP Unique ID	n/a
Structure Solved
Class	Alpha+Beta
Molecularity	Monomer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	259
Molecular Weight	29014.7
Pi	6.13
Molecular Weight	29014.7
Disulphides	0
Full Sequence	MGSSHHHHHHSSGLVPRGSHMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEGVTAAGITLGMDELYK
Notes	n/a

Expression
Report	Cabanne, C., Noubhani, A. M., Hocquellet, A., Dole, F., Dieryck, W., Santarelli, X. (2005) J Chromatography B, 818, 23-27
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	C41
Expression Temp	30.0
Expression Time	15
Expression Vector	pET15b
Expression Protocol	Cells were grown at 37degC in 1.5 L of LB medium containing 100microg/ml ampicillin and 10microg/ml tetracycline in a bioreactor. Incubated, induced cells were harvested by centrifugation and the resulting pellet resuspended in 40ml cold 20mM sodium bicarbonate, pH 9.0 and sonicated.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.6
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Ion-exchange chromatography
Solubility	insoluble

Refolding
Refolding Method	column refolding: ion-exchange chromatography
Wash Buffer	n/a
Solubilization Buffer	8M urea, 20mM sodium bicarbonate, pH 9.0
Refolding Buffer	20mM sodium bicarbonate, pH 9.0
Pre-Refolding Purification	Ion-exchange chromatography
Tag Cleaved	no
Refolding pH	9.0
Refolding Temperature	20.0
Protein Concentration	1.25mg/ml
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Homogenate was diluted with 360ml of solubilization buffer, and agitated for 2 hours at 15degC. Suspension underwent anion exchange expansion using a Streamline 25 column. This was preformed using solubilization buffer applied at 44ml/min. After a run of two volumns refolding was preformed with 10 columns worth of refolding buffer applied at the same rate. Elution was preformed in two steps, the EGFP using 150mM NaCl, 20mM sodium bicarbonate, pH 9.0 and 1 M NaCl, 50mM TrisCl, pH 8.0 for the elution of contaminates.
Refolding Assay	Fluorescence
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a