Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Low density lipoprotein receptor
Abbreviated Name	LDLR
SCOP Family	LDL receptor-like module
Structure Notes
Organism	Human
UniProt Accession	P01130
SCOP Unique ID	n/a
Structure Solved
Class	Small Proteins
Molecularity	Unknown

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	294
Molecular Weight	32442.5
Pi	4.32079
Molecular Weight	32442.5
Disulphides	>10
Full Sequence	GS AVGDRCERN EFQCQDGKCI SYKWVCDGSA ECQDGSDESQ ETCLSVTCKS GDFSCGGRVN RCIPQFWRCD GQVDCDNGSD EQGCPPKTCS QDEFRCHDGK CISRQFVCDS DRDCLDGSDE ASCPVLTCGP ASFQCNSSTC IPQLWACDND PDCEDGSDEW PQRCRGLYVF QGDSSPCSAF EFHCLSGECI HSSWRCDGGP DCKDKSDEEN CAVATCRPDE FQCSDGNCIH GSRQCDREYD CKDMSDEVGC VNVTLCEGPN KFKCHSGECI TLDKVCNMAR DCRDWSDEPI KEC
Notes	n/a

Expression
Report	Simmons T, Newhouse YM, Arnold KS, Innerarity TL, Weisgraber KH (1997) J Biol Chem, 272, 25531-25536
Project Aim	Recombinant Protein Expression
Fusion	N-terminal thioredoxin + hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)
Expression Temp	37.0
Expression Time	2h
Expression Vector	pET32a
Expression Protocol	1L cultures in LB broth were inoculated with 50ml overnight culture, cells were grown to mid-log phase and expression was induced with 0.5mM IPTG. Cells were grown a further 2h at 37degC and harvested by centrifugation (4000rpm, 20min).
Method of Induction	IPTG
Cell Density at Induction	OD 550 = 0.6
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Affinity chromotography
Solubility	soluble

Refolding
Refolding Method	Dilution/Dialysis combination
Wash Buffer	n/a
Solubilization Buffer	25mM Tris pH 7.5, 2.5mM EDTA, 7M GdnHCl, 1% 2-mercaptoethanol
Refolding Buffer	1:20mM glycylclycine pH 9.0, 20mM CaCl2, 10mM cystemine, 1mM cstamine 2: 20mM glycylclycine pH 9.0, 20mM CaCl2
Pre-Refolding Purification	Affinity chromotography
Tag Cleaved	yes
Refolding pH	9.0
Refolding Temperature	4.0
Protein Concentration	60microg/ml
Refolding Time	n/a
Redox Agent	cysteamine/cystamine
Redox Agent Concentration	10mM/1mM,10mM/1mM
Refolding Protocol	Cell pellets were resuspended in 1% of the original volume of lysis buffer (25mM Tris pH 7.5, 2.5mM EDTA), lysed by sonication and centrifuged (18000g, 20min). The cleared lysate was incubated at 80degC for 7 min with constant agitation. The precipiate was removed by centrifugation (20min, 18000g) and the receptor fusion in the supernatant was cleaved by incubation with thrombin overnight at room temperature at a weigt ration of 1:100. 7M GdnHCl and 1% 2-mercaptoethanol were added to the cleaved mixture, which was then incubated for 4h at 4degC and loaded onto a Sephacryl S-300 HR column, protein was eluted with 4M GdnHCl, 100mM Tris pH 7.4, 1mM EDTA, 0.1% 2-mercaptoethanol. Selected fractions were pooled and dialyzed against 4M GdnHcl at 4degC under argon to remove the 2-mercaptoethanol. The protein was refolded and oxidized by dilution to approx 60microg/ml in refolding buffer 1 and dialysed against refolding buffer 2, and then purified by affinity chromatography
Refolding Assay	Bioactivity,SDS-PAGE,Isoelectic focusing
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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