Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Interleukin-1 receptor antagonist protein
Abbreviated Name	IL-1ra
SCOP Family	Interleukin-1 (IL-1)
Structure Notes
Organism	Human
UniProt Accession	P18510
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Unknown

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	152
Molecular Weight	17126.4
Pi	5.46
Molecular Weight	17126.4
Disulphides	1
Full Sequence	RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE
Notes	n/a

Expression
Report	Seefeldt MB, Crouch C, Kendrick B, Randolph TW. (2007) Biotechnology and Bioengineering, 98, 476-485
Project Aim	Protein refolding
Fusion	None
Protein Expression and Production	Protein expressed and purified in native conformation prior to denaturation and refolding.
Expression Host	None
Expression Strain	None
Expression Temp	0.0
Expression Time	0
Expression Vector
Expression Protocol
Method of Induction	Not Stated
Cell Density at Induction	OD =
Cell Disruption Method	None
Lytic Agent	None
Pre-Refolding Purification	None
Solubility

Refolding
Refolding Method	High Pressure
Wash Buffer	10 mM sodium citrate,pH 6.5, 0.5 mM EDTA, and 140 mM NaCl
Solubilization Buffer	20 mM Tris (pH 7.0 at 31C), 100 mM NaCl and 10 mM b-mercaptoethanol
Refolding Buffer	20 mM Tris (pH 7.0 at 31C), 100 mM NaCl and 10 mM hydrogen peroxide
Pre-Refolding Purification	None
Tag Cleaved	no
Refolding pH	7.0
Refolding Temperature	31.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a,n/a,n/a,n/a
Refolding Protocol	Thermally induced and pressure-induced precipitates of IL- 1ra were washed with reducing buffer, the aggregate allowed to settle, and the supernatant removed. This process was repeated three times to minimize the amount of unaggregated IL-1ra remaining in the precipitate. The aggregates were resuspended in 600 mL of reducing buffer at an IL-1ra concentration of approximately 5.5 mg/mL and placed in heat-sealed 1 mL polypropylene syringes. Samples were pressurized slowly over 10 min to minimize system heating and then incubated at 318C for 14 h (overnight) at pressures of 1,500 or 1 bar (atmospheric control). Previous studies have demonstrated that 14 h incubation times are adequate refolding times for many proteins (Kim et al., 2006). The samples were slowly depressurized over 10 min. After depressurization, the samples were centrifuged and concentration of soluble protein was measured by absorbance. Aggregate concentration was measured by dissolving the precipitate in 750 mL of 8 M guanidine HCl prior to extinction coefficient analysis. Solubilization yields were determined by dividing the monomer mass fraction by the total protein concentration (monomer and aggregate) and then converting the result to a percentage. SEC-HPLC and activity assays were conducted to verify activity and the proper size of the protein after pressure-based refolding of thermally induced aggregates, confirming the formation of native structure. SEC-HPLC and activity assays were not performed after the initial solubilization measurements after pressure-based solubilization of pressure-induced aggregates since refolding was ongoing at atmospheric pressure, confounding results.
Refolding Assay	Activity assay,SEC-HPLC
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	57%
Purity	n/a
Notes	n/a

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