Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Interleukin-7
Abbreviated Name	IL-7
SCOP Family	Unknown
Structure Notes
Organism	Human
UniProt Accession	P13232
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	152
Molecular Weight	17387.2
Pi	8.71
Molecular Weight	17387.2
Disulphides	Unknown
Full Sequence	DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH
Notes	n/a

Expression
Report	Ouellette T, Destrau S, Ouellette T, Zhu J, Roach JM, Coffman JD, Hecht T, Lynch JE, Giardina SL. (2003) Protein Expression and Purification, 30, 156-66
Project Aim	Protein refolding
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	E coli
Expression Strain	HMS174 (DE3) pLysS
Expression Temp	37.0
Expression Time	3 h
Expression Vector	pET-24a
Expression Protocol	The resulting expression vector pLC124 is kanamycin-resistant. The expression system HMS174 (DE3) pLysS The fermenter was maintained at a temperature of 37 °C with an agitation rate of 150 rpm increasing to 350 rpm, an aeration rate of 2.5–9 scfm, and a vessel pressure of 5 psig. The %DO and its maintenance were identical to the first-stage fermenter. The fermenter was aseptically inoculated with 11 L (1.5%, v/v) of the first-stage fermenter. The culture was induced with filter- sterilized, dioxane-free IPTG at a 1-mM final concentration when the OD600 reached 9.5 ( +_2.0). The culture was prepared for harvest 3 h after IPTG induction. The cell paste was recovered, using two tubular bowl centrifuges (Sharples AS16) in parallel, with a speed of 16,500 rpm. The fermentation yielded 9.3 kg of cell paste that was divided into approximately 1 kg aliquots and stored at )70 to )90 °C until proceeding to the inclusion body recovery phase of the process.
Method of Induction	IPTG
Cell Density at Induction	OD 9.5 = 600
Cell Disruption Method	High pressure homogenization
Lytic Agent	None
Pre-Refolding Purification	Size-exclusion chromatography
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	50 mM sodium phosphate, pH 7.4, 100 mM sodium chloride, and 20 mM EDTA at a ratio of 2.33 L of buffer per kg
Solubilization Buffer	6 M guanidine HCl
Refolding Buffer	0.1 M Tris, 0.5 M L-arginine and 2 mM EDTA, pH 9
Pre-Refolding Purification	Size-exclusion chromatography
Tag Cleaved	no tag
Refolding pH	9.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	16-18 h
Redox Agent	GSSG
Redox Agent Concentration	0.55 g/l
Refolding Protocol	The refolding conditions consisted of a 1:100 dilution of the Superdex 200 purified denatured rhIL-7 to a final protein concentration of 80–100 lg/mL. A 240.0-mL aliquot (2.4 g) of denatured Superdex 200 purified rhIL-7 was reduced with 10 mg DTE per mL for 16–18 h at room temperature. Oxidized glutathione was added to 25 L of refolding buffer (0.1 M Tris, 0.5 M L L -arginine, and 2 mM EDTA, pH 9) to a final concentration of 0.55 g/L and mixed thoroughly with an overhead impeller. The reduced rhIL-7 solution was added to the completed refold buffer with a Mannostat pump at 110 mL/min with continuous mixing. The solution was incubated without mixing at 2–8 °C for 48–72 h before proceeding to the HIC chromatography step.
Refolding Assay	SDS-PAGE,RP-HPLC
Refolding Chaperones	None
Refolding Additives	L-Arginine
Additives Concentration	0.5 M
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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