Refold - P-Glycoprotein

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	P-Glycoprotein
Abbreviated Name	Human
SCOP Family	Unknown
Structure Notes
Organism	Human
UniProt Accession	P08183
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Monomer

Construct
Full Length	n
Domain	N-Terminal Nucleotide Binding Domain Mutant
Chimera	n/a
Variants	n/a
Chain Length	233
Molecular Weight	25735.5
Pi	9.52
Molecular Weight	25735.5
Disulphides	0
Full Sequence	ARGAAYEIFKIIDNKPSIDSYSKSGHKPDNIKGNLEFRNVHFSYPSRKEVKILKGLNLKVQSGQTVALVGNSGAGKSTTVQLMQRLYDPTEGMVSVDGQDIRTINVRFLREIIGVVSQEPVLFATTIAENIRYGRENVTMDEIEKAVKEANAYDFIMKLPHKFDTLVGERGAQLSGGQKQRIAIARALVRNPKILLLDEATSALDTESEAVVQVALDKARKGRTTIVIAHRLS
Notes	pET-NBD1MDR1-C431A (358-590)

Expression
Report	Booth, C., Pulaski, L., Gottesman, M. M., Pastan, I. (2000) Biochemistry, 39, 5518-5526
Project Aim	Structure-Function
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(lambdaDE3)
Expression Temp	37.0
Expression Time	90min
Expression Vector	pET3a
Expression Protocol	Cells were grown overnight on LB agar containing 100micgrog/ml ampicillin and superbroth (2% glucose, 0.05% MgSO4). After induction cells were harvested via centrifugation(3500rpm, 30min) Pellet was washed by sequential resuspension and centrifugation(13,000rpm, 50min) in 200ml of buffer A(50mM Tris and 20mM EDTA, pH 8.0). During initial washes buffer A contained 2.5% Triton X-600, this was then decreased to 1%, prior to three washes in the absence of detergent. The final pellet was resuspended in 20ml of buffer A and then centrifuged(20,000rpm, 20min) to obtain inclusion bodies.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 3
Cell Disruption Method	Not stated
Lytic Agent	Detergents
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dilution/Dialysis combination
Wash Buffer	n/a
Solubilization Buffer	6M GuHCl, 0.1M Tris, 2mM EDTA, pH 8.0
Refolding Buffer	0.1M Tris, 0.5M arginine, 2mM EDTA, pH 8.0
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	10.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Inclusion body were solubilized and centrifuged(20,000rpm, 20min) and stored at room temp for at least 2 hours. Solubilized inclusion bodies were harvested by centrifugation(20,000rpm, 30min) before renaturation. Refolding was preformed by a rapid 100 fold dilution in refolding buffer, this was maintained at 10degC for 12hrs, before overnight dialysis against 20mM Tris, pH 8.0/ 100mM NaCl.
Refolding Assay	Far-UV Circular Dichroism,Enzyme activity,SDS-PAGE
Refolding Chaperones	None
Refolding Additives	L-Arginine,Glycerol,Triton X-100
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a