Refolding Record:

Protein See all refolding records for this protein...
Protein Name	HRP/Fatty acid-binding protein, heart fusion
Abbreviated Name	HRP-FABP
SCOP Family	Fatty acid binding protein-like
Structure Notes
Organism	Human
UniProt Accession	P05413
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	132
Molecular Weight	14726.8
Pi	6.33
Molecular Weight	14726.8
Disulphides	Unknown
Full Sequence	VDAFLGTWKLVDSKNFDDYMKSLGVGFATRQVASMTKPTTIIEKNGDILTLKTHSTFKNTEISFKLGVEFDETTADDRKVKSIVTLDGGKLVHLQKWDGQETTLVRELIDGKLILTLTHGTAVCTRTYEKEA
Notes	n/a

Expression
Report	Grigorenko V, Andreeva I, Börchers T, Spener F, Egorov A. (2001) Anal Chem., 73, 1134-1139
Project Aim	Undefined
Fusion	C-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)pLysS
Expression Temp	30.0
Expression Time	3 h
Expression Vector	pETHRP-FABPhis
Expression Protocol	Expression, Refolding, and Purification of Recombinant HRP-FABPhis Conjugate. E. coli BL21(DE3)pLysS bacteria were transformed with pETHRP-FABPhis (Figure 1) and grown under vigorous shaking at 37 °C in 300 mL LB medium containing 100 µg/mL ampicillin, 34 µg/mL chloramphenicol to an OD550 of 0.6 and induced with 0.4 mM IPTG. Bacteria were further grown at 30 °C for 3 h, harvested by centrifugation (3600g, 15 min, 4 °C) and lysed by freezing at -70 °C, thawing in 10 mL of buffer A (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 10 mM DTT), and further incubating on ice for 30 min. Lysis was completed by sonication with three 30-s pulses (Branson onifier).
Method of Induction	IPTG
Cell Density at Induction	OD 0.6 = 550
Cell Disruption Method	Freeze/Thaw+Sonication
Lytic Agent	None
Pre-Refolding Purification	not specified
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	50 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 10 mM DTT, 1% Triton X-100.
Solubilization Buffer	50 mM Tris-HCl, 6 M urea, pH 8.0, and 2 mM DTT
Refolding Buffer	20 mM Tris-HCl, 1.7 M urea, 4% glycerol, pH 8.5, and 2 mM CaCl2, 0.7 mM GSSG
Pre-Refolding Purification	not specified
Tag Cleaved	no
Refolding pH	8.5
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	overnight
Redox Agent	GSSG
Redox Agent Concentration	0.7 mM
Refolding Protocol	The solubilized inclusion bodies (5 mL) were added drop-by-drop to 95 mL of refolding medium (20 mM Tris-HCl, 1.7 M urea, 4% glycerol, pH 8.5, and 2 mM CaCl2). Thereafter, oxidized L-glutathione was added to a final concentration of 0.7 mM, and the mixture was left overnight at 4 °C without stirring. The solution now containing refolded apoHRP-FABPhis was stirred with 5 mL Ni-NTA agarose (Qiagen, Hilden, Germany) for 2 h at 4 °C. The Ni-NTA agarose was collected in a column (1.5 cm diameter) and bound apoHRP-FABPhis was eluted at room temperature using an imidazole-containing buffer (10 mM Tris-HCl, 0.15 M NaCl, 2 M urea, 200 mM imidazole, pH 7.4). Fractions containing apoHRP-FABPhis were collected, dialyzed against PBS (10 mM Na-phosphate, 0.15 M NaCl, pH 7.4) and reconstituted with hemin, which was added drop-by-drop from an ∼1 mM stock solution in 0.01 M KOH. The final concentration of the added hemin did not exceed 3 times the concentration of apoHRP-FABPhis, as estimated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The sample was concentrated by ultrafiltration and passed through a column with Sephadex G-25 (1.5 × 25 cm) equilibrated with PBS to remove unbound hemin.
Refolding Assay	Enzyme activity
Refolding Chaperones	None
Refolding Additives	Glycerol
Additives Concentration	4%
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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