Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Interferon gamma
Abbreviated Name	IFN gamma
SCOP Family	Interferons/interleukin-10 (IL-10)
Structure Notes
Organism	Human
UniProt Accession	P01579
SCOP Unique ID	n/a
Structure Solved
Class	Alpha
Molecularity	Dimer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	146
Molecular Weight	17145.6
Pi	9.53939
Molecular Weight	17145.6
Disulphides	0
Full Sequence	CYCQDPYVKE AENLKKYFNA GHSDVADNGT LFLGILKNWK EESDRKIMQS QIVSFYFKLF KNFKDDQSIQ KSVETIKEDM NVKFFNSNKK KRDDFEKLTN YSVTDLNVQR KAIHELIQVM AELSPAAKTG KRKRSQMLFR GRRASQ
Notes	n/a

Expression
Report	Gao YG, Guan YX, Yao SJ, Cho MG (2003) Biotechnol Prog, 19, 915-920
Project Aim	Undefined
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	None
Expression Temp	42.0
Expression Time	6h
Expression Vector	pBV220
Expression Protocol	2ml of overnight seed culture grown at 30degC was transferred to 200ml of 5xM9+LB medium (per L:15g tryptone, 7.5g yeast, 5g NaCl, 6g Na2HPO4, 3g KH2PO4, 1g NH4Cl, 3mg CaCL2 with 1mL x 1M MgSO4.7H2O and 10ml of 20% sucrose added before use) with 0.1mg/ml ampicillin present. Cells were incubated at 30degC until OD600 reached 0.8, when expression was induced by shifting the temperature to 42degC with incubation for a further 6h.
Method of Induction	Temperature Shift
Cell Density at Induction	OD 600 = 0.8
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Chaperone-assisted refolding
Wash Buffer	0.05M TrisHCl pH 8.0, 1.5M urea, 0.4% Triton-X100, 10mM EDTA
Solubilization Buffer	0.05M TrisHCl pH 8.0, 6M GdnCl, 0.01M DTT
Refolding Buffer	0.05M PBS pH 7.0, 5mM EDTA, 20mM KCl, 6.3/63/126 microM mini-GroEL, 0.5/1/1.5/2M urea
Pre-Refolding Purification	None
Tag Cleaved	yes
Refolding pH	7.0
Refolding Temperature	25.0
Protein Concentration
Refolding Time
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Cells were harvested by centrifugation, then cells were resuspended in 200mL buffer A (0.05M TrisHCl pH 8.0) and centrifuged again. The cells were then resuspended 10 volumes of buffer A and sonicated for 100 cycles of 5sec-on, 3-sec off. Following centrifugation (6000g x 15min, 4degC) the inclusion bodies were resuspended in wash buffer and incubated at 30min for 37degC. The mixture was then centrifuged again. The inclusion bodies were incubated in 10ml of solubilization buffer for 90 min at 37degC with agitation at 120rpm. After centrifugation the supernatant was retained. A mini-GroEL construct (a.a.191-345 of GroEL, functional fragment) expressed and purified with a hexahistidine affinity tag was mixed with Ni-NTA resin for 1hr at a coupling density of 2.1mg/ml resin. 12ml of mini-GroEL-linked resin was packed into a column, then 1cm of Sephadex G200 was packed at the top of the column. The column was then equilibrated with refolding buffer. The denatured protein was then applied to the column and was eluted with refoling buffer. The eluted protein was then dialyzed against 0.05M Tris pH 8.0
Refolding Assay	Bioactivity
Refolding Chaperones	None,GroEL minichaperone
Refolding Additives	None
Additives Concentration
Refolding Yield
Purity
Notes	Protein could also be refolded by dilution into 60x refolding buffer at 25degC, but did not allow for re-use of mini-GroEL

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