Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Beta-2-microglobulin
Abbreviated Name	Beta2m
SCOP Family	C1 set domains (antibody constant domain-like)
Structure Notes
Organism	Human
UniProt Accession	P61769
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Monomer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	101
Molecular Weight	11731.2
Pi	6.07
Molecular Weight	11731.2
Disulphides	Unknown
Full Sequence	IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM
Notes	n/a

Expression
Report	Ferré H, Ruffet E, Nielsen LL, Nissen MH, Hobley TJ, Thomas OR, Buus S. (2005) Protein Science, 14, 2141-53
Project Aim	Protein refolding
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	None
Expression Temp	0.0
Expression Time	00
Expression Vector	pET28a
Expression Protocol	he construct comprising MG-HAT-GS-FXa-hβ2m was excised using NcoI and HindIII and inserted into the pET28a+ vector (Novagen) containing the kanamycin resistance gene, and subsequently transformed into E. coli strain BL21(DE3). Clones, which produced protein upon induction with IPTG, were identified and the inserted gene sequence was verified by DNA sequencing (ABI310, Perkin Elmer). Expression was done in a 2-L Labfors fermentor (Infors) by IPTGinduction as previously described by Ferré et al. (2003).
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Not stated
Lytic Agent	Lysozyme
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dilution and expand bed absorption (EBA)
Wash Buffer	8 M urea, 20 mM Tris- HCl (pH 8.0)
Solubilization Buffer	8 M urea, 20 mM Tris- HCl (pH 8.0)
Refolding Buffer	20 mM Tris-Hcl [pH 8.0]
Pre-Refolding Purification	None
Tag Cleaved	no
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Batch refolding Analytical-scale batch refolding was performed in triplicate by dilution of denatured feedstocks with refolding buffer (20 mM Tris-Hcl [pH 8.0]) in 2-mL Eppendorf tubes in a total reaction volume of 1 mL. Experiments at 10 μg/mL were performed in a 2-mL reaction volume to provide enough protein for the subsequent analysis. Each reaction mixture was incubated for 0.5 h at room temperature (RT) on a vertical rotating mixer. Insoluble aggregates were removed by centrifugation at 15,000g for 10 min at 4°C, and the resulting supernatants were then analyzed for total soluble protein and purity using a BCA protein assay (see below) and SDS-PAGE, respectively. Preparative batch refolding was initiated by adding 20 mL denatured feedstock into 700 mL binding buffer, resulting in a final urea concentration of 222 mM. The reaction was stirred using a magnetic rod (200 rpm) for 0.5 h at RT before the suspension was loaded onto the EBA column.
Refolding Assay	Peptide-MHC -I binding assay
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	45%
Purity	n/a
Notes	n/a

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