Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Plasma retinol-binding protein
Abbreviated Name	RBP
SCOP Family	Retinol binding protein-like
Structure Notes
Organism	Human
UniProt Accession	P02753
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	183
Molecular Weight	21071.6
Pi	5.27
Molecular Weight	21071.6
Disulphides	3
Full Sequence	ERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRSERNLL
Notes	n/a

Expression
Report	Folli C, Viglione S, Busconi M, Berni R. (2005) Biochemical and Biophysical Research Com, 336, 1017-1022
Project Aim	Diagnostic studies
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	Origami B(DE3)
Expression Temp	37.0
Expression Time	overnight
Expression Vector	pET11b
Expression Protocol	BQ645928 corresponding to mature RBP (552 bp) was PCR-amplified using the NdeI-tailed upstream primer 5′-ATACATATGGAGCGCGACTGCCGAGTG-3′ and the BamHI-tailed downstream primer 5′-TAAGGATCCGATTCTTGATATTGCTACAAAAGG-3′. The amplification product was inserted into the pGEM vector (Promega). The restriction fragment obtained from NdeI/BamHI digestion of plasmid pGEM-RBP was then ligated into the dephosphorylated NdeI and BamHI sites of the expression vector pET11b (Novagen), and the resulting plasmid (pET-RBP) was electroporated into Escherichia coli Origami (DE3) cells (Stratagene). The expression of human RBP was induced by 1 mM isopropyl-1-thio-β-d-galactopyranoside, and, after an overnight incubation at 37 °C, cells were lysed by sonication.
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	not specified
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	n/a
Solubilization Buffer	Tris–HCl 50 mM (pH 7.5), EDTA 2 mM, PMSF 2 mM, Triton X-100 0.1% (V/V), and urea 8 M)
Refolding Buffer	Tris–HCl 50 mM (pH 9.3)
Pre-Refolding Purification	not specified
Tag Cleaved	no tag
Refolding pH	9.3
Refolding Temperature	0.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	The recombinant RBP, which was present in the insoluble fraction, was dissolved in a denaturing medium (Tris–HCl 50 mM (pH 7.5), EDTA 2 mM, PMSF 2 mM, Triton X-100 0.1% (V/V), and urea 8 M) and then refolded by dialysis against Tris–HCl 50 mM (pH 9.3). Human RBP was then purified to homogeneity by using a two-step procedure, comprising gel filtration chromatography (Bio-Gel P-60, Bio-Rad) and affinity chromatography on a human TTR-Sepharose 4B affinity column, prepared by coupling human TTR to CNBr-activated Sepharose 4B (Pharmacia) according to [20]. Regarding this latter step, the proteins applied to the affinity column lacking affinity for TTR were eluted from the column in the presence of the equilibrating buffer (sodium phosphate 0.05 M (pH 7.4), NaCl 0.15 M), whereas correctly folded RBP possessing affinity for TTR was retained and could be obtained from the column in one single elution step at low ionic strength (sodium phosphate 0.001 M (pH 7)). The final yield was approximately 20 mg/liter of cell culture. Protein concentrations were determined by using absorption coefficients (A 1 mg/ml, 1 cm) at 279 nm of 1.74 and 2.02 for apo- and holo-RBP, respectively, and 1.43 for TTR [11].
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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