Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Resistin
Abbreviated Name	Resistin
SCOP Family	Resistin (Pfam 06954)
Structure Notes
Organism	Mouse
UniProt Accession	RSN_MOUSE
SCOP Unique ID	n/a
Structure Solved
Class	Small Proteins
Molecularity	Monomer

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	107
Molecular Weight	11738.4
Pi	8.03356
Molecular Weight	11738.4
Disulphides	5
Full Sequence	MRGSHHHHHH TDP SSMPLCPIDE AIDKKIKQDF NSLFPNAIKN IGLNCWTVSS RGKLASCPEG TAVLSCSCGS ACGSWDIREE KVCHCQCARI DWTAARCCKL QVAS
Notes	One inter-chain disulfide bond formed between molecules

Expression
Report	Juan CC, Kan LS, Huang CC, Chen SS, Ho LT, Au LC (2003) J Biotechnology, 103, 113-117
Project Aim	Structure-Function
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	JM109
Expression Temp	37.0
Expression Time	6h
Expression Vector	pQE31
Expression Protocol	Cells were grown at 37degC in 600ml 2xTY broth with ampicillin. At log phase, 1mM IPTG and 1.5mM cysteine were added and cells were incubated a further 6h and then harvested by centrifugation and frozen overnight.
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	25mM TrisHCl, 50mM NaCl, 0.5% Tween 20, 0.1mM EDTA pH 7.6
Solubilization Buffer	6M GdnHCl, 20mM TrisHCl, 8mM 2-mercaptoethanol pH 7.5
Refolding Buffer	1:40mM TrisHCl, 0.1mM DTT, 0.1% mannitol pH 11, 2:40mM TrisHCl, 0.1mM DTT, 0.1% mannitol pH 8.8, 3:40mM TrisHCl pH 8.8, 4:10mM KH2PO4 pH 8.5
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no
Refolding pH	8.8
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	DTT
Redox Agent Concentration	0.1mM,0.1mM
Refolding Protocol	The cell pellet was thawed and resuspended in 50mM NaCl, 25mM TrisHCl pH 7.6, 150microg/ml DNaseI and then sonicated. The lysate was centrifuged and the pellet was resuspended in wash bffer and incubated on ice for 10min. The inclusion bodies were collected by centrifugation (4degC, 10000K, 15min), then the pellet was dissolved in solubilization buffer and incubated at 37degC for 1h. The solution was then passed through a 5ml Ni-NTA column. The column was washed with solubilization buffer, followed by 6M GdnHCl 50mM sodium acetate, 8mM 2-mercaptoethanol pH 6.5, and then 6M GdnHCl, 50mM sodium acetate pH 5.9. The protein was then eluted in 4.5M urea, 50mM sodium acetate pH 4.5. The pH of the eluted protein solution was adjusted to 11 by Tris base, and 10mM DTT and 0.1% manitol were added. The solution was then incubated at 37degC for 1h. Refolding was performed by serial dialysis in a range of buffers: firstly, in 3 changes refolding buffer 1, each time with decreasing amounts of urea present (2,1,0M), followed by refolding buffers 2, 3, and 4.
Refolding Assay	Far-UV Circular Dichroism,Bioactivity,Western Blot,SDS-PAGE
Refolding Chaperones	None
Refolding Additives	None,Mannitol
Additives Concentration	0.1%
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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