Refold - Anti-human ferritin antibody F11 VL domain

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Anti-human ferritin antibody F11 VL domain
Abbreviated Name	VL domain
SCOP Family	Unknown
Structure Notes
Organism	Human
UniProt Accession
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Unknown

Expression
Report	Tsybovsky Y, Shubenok DV, Kravchuk ZI, Martsev SP. (2007) Protein Eng Des Sel., 20, 481-490
Project Aim	Folding
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	37.0
Expression Time	3 h
Expression Vector	pETVL
Expression Protocol	Purification of the VL domain Expression of the VL domain was performed as we described earlier (Martsev et al., 2000). Inclusion bodies, which contained the major (95%) fraction of the recombinant protein, were separated from the soluble fraction of the cell lysate by centrifugation for 30 min at 35 000 g.
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Metal affinity chromatography
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	150-200 mL of 50 mM Tris-HCl, pH 8.0, containing 0.15 M NaCl and 1 mM phenylmethanesulfonyl fluoride (PMSF)
Solubilization Buffer	6 M guanidine hydrochloride (Gdn–HCl)
Refolding Buffer	5, 3 and 1 M urea in 0.1 M Tris, pH 8.1, or 0.1 M sodium phosphate, pH 7 and, finally, against 0.1 M sodium phosphate, pH 7.4
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no
Refolding pH	7.4
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	To obtain the VL-2 conformer, we applied the refolding protocol-2 (Fig. 1, bottom line). For this, the VL domain after the Ni2+-NTA chromatography was dialyzed against 5, 3 and 1 M urea in 0.1 M Tris, pH 8.1, or 0.1 M sodium phosphate, pH 7 and, finally, against 0.1 M sodium phosphate, pH 7.4, without the denaturant.
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	In this paper, we provide the first description of the two stable and functional conformations of an antibody domain The key difference between the protocols is exposure to low pH (pH 2–3) in refolding protocol-1, which was critical to obtain the conformation denoted as VL-1. When exposure to the low pH was excluded from the procedure to give the refolding protocol-2, the stepwise dialysis at near-neutral pH yielded the conformer VL-2