Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Dihydrolipoamide dehydrogenase
Abbreviated Name	DHLipDH
SCOP Family	FAD/NAD-linked reductases, N-terminal and central domains
Structure Notes
Organism	Haloferax volcanii
UniProt Accession	Q04829
SCOP Unique ID	n/a
Structure Solved
Class	Alpha/Beta
Molecularity	14-mer

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	496
Molecular Weight	52458.7
Pi	4.36565
Molecular Weight	52458.7
Disulphides	Unknown
Full Sequence	MVVGDIATGTE LLVIGAGPGG YVAAIRAAQN GIDTTLVEKD AYGGTCLNYG CIPSKALITG ANLAHEAGNA EEMGIHADPV VDMSQLRDWK SGVVDQLTGG VEKLCKANGV NLVEGTARFK DENAVRIAHG GEGQGSETIE FEHCIIATGS RVIQIPGFDF GDEPVWSSRD ALEADTVPER LVVVGGGYIG MELSTTFAKL GADVTVVEML DDILPGYESD VARVVRKRAE ELGIDMHLGE GATGWREEDD GIMVTTETED GEENEYRADK VLVAVGRSPV TDTMDIENAG LEADDRGFLS VDDRRRTDVE HIYAVGDVVE DTPMLAHVAS KEGIVAAEHV AGEPVAFDSQ AVPAAVFTDP EIGTVGMTEA DAEEAGFTPV VGQMPFRASG RALTTNHADG FVRVVADEES GFVLGAQIVG PEASELIAEL AFAIEMGATL EDVASTIHTH PTLAEAVMEA AENALGQAIH TLNR IRLLTKPERKLSWLLPPLSNN
Notes	n/a

Expression
Report	Connaris H, Chaudhuri JB, Danson MJ, Hough DW (1999) Biotechnology and Bioengineering, 64, 38-45
Project Aim	Recombinant Protein Expression
Fusion	None
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	3h
Expression Vector	pET3d
Expression Protocol	Cells were grown in LB medium containing 50microg ampicillin/ml. When OD 600 reached 0.6, expression was induced with 0.4mM IPTG. Cells were grown a further 3h before harvesting by centrifugation.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.6
Cell Disruption Method	Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	Washing inclusion body
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	20mM TrisHCl pH 8, 2M KCl, 2mM EDTA
Solubilization Buffer	20mM TrisHCl pH 8, 2mM EDTA, 8M uirea, 50mM DTT
Refolding Buffer	20mM TrisHCl pH 8, 2M KCl, 2mM EDTA, 10microM FAD 10mM NAD, 0.3mM GSSG, 3mM GSH
Pre-Refolding Purification	Washing inclusion body
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	24h+
Redox Agent	GSH/GSSG
Redox Agent Concentration	3mM/0.3mM
Refolding Protocol	The cell pellets were resuspended in 20mM TrisHCl pH 8 containing 2mM EDTA and 2M KCl. Cells were treated with 100microg/ml lysozyme and 0.1 volume of 1% Triton-X100 and incubated at 30degC for 1h before being transferred onto ice for 15min. The suspension was sonicated and centrifuged (10000g, 10min, 4degC). The pellet was washed twice in wash buffer and centrifuged, then dissolved in solubilization buffer. Refolding was performed by slowly diluting the protein 20-fold into refolding buffer and incubating for at least 24h at 4degC. The refolded protein was dialysed at 4degC against 2L 50mM potassium phosphate pH 7.2, 200mM NaCl. The protein was then centrifuged and the supernatant was purified using a metal ion affinity column. b
Refolding Assay	SDS-PAGE,Bradford assay
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	Different concentrations of FAD, NAD, GSSG, GSH tested for optimal refolding recovery, FAD is necessary for renaturation. Optimal refolding after 6days. Protein solubilised in guandine-containing buffer did not refold.

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