Refolding Record:

Protein See all refolding records for this protein...
Protein Name	UvrA Protein
Abbreviated Name	UvrA Protein
SCOP Family	Unknown
Structure Notes
Organism	Escherichia coli
UniProt Accession	P0A698
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Unknown

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	680
Molecular Weight	75155.8
Pi	6.26594
Molecular Weight	75155.8
Disulphides	0
Full Sequence	MDKIEVRGAR THNLKNINLV IPRDKLIVVT GLSGSGKSSL AFDTLYAEGQ RRYVESLSAY ARQFLSLMEK PDVDHIEGLS PAISIEQKST SHNPRSTVGT ITEIHDYLRL LFARVGEPRC PDHDVPLAAQ TVSQMVDNVL SQPEGKRLML LAPIIKERKG EHTKTLENLA SQGYIRARID GEVCDLSDPP KLELQKKHTI EVVVDRFKVR DDLTQRLAES FETALELSGG TAVVADMDDP KAEELLFSAN FACPICGYSM RELEPRLFSF NNPAGACPTC DGLGVQQYFD PDRVIQNPEL SLAGGAIRGW DRRNFYYFQM LKSLADHYKF DVEAPWGSLS ANVHKVVLYG SGKENIEFKY MNDRGDTSIR RHPFEGVLHN MERRYKETES SAVREELAKF ISNRPCASCE GTRLRREARH VYVENTPLPA ISDMSIGHAM EFFNNLKLAG QRAKIAEKIL KEIGDRLKFL VNVGLNYLTL SRSAETLSGG EAQRIRLASQ IGAGLVGVMY VLDEPSIGLH QRDNERLLGT LIHLRDLGNT VIVVEHDEDA IRAADHVIDI GPGAGVHGGE VVAEGPLEAI MAVPESLTGQ YMSGKRKIEV PKKRVPANPE KVLKLTGARG NNLKDVTLTL PVGLFTCITG VSGSGKSTLI NDTLFPIAQR QLNGATIAEP APYRDIQGLE
Notes	C-terminal deletion mutant, lacking the 260 residues from the C-terminus

Expression
Report	Claassen LA, Ahn B, Koo HS, Grossman L. (1991) J Biol Chem, 266, 11380-11387
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	HB101
Expression Temp	42.0
Expression Time	2h
Expression Vector	pLAC1
Expression Protocol	500ml cultures were grown in LB broth with 50microg/ml ampicillin at 30degC until A600 reached 1.0. Expression was induced by the addition of 500ml LB broth which had been pre-heated to 54degC, the diluted culture was then incubated for 2h at 42degC. The culrutes were harvested by centrifugation (6000rpm, 15min, 4degC) and the cell pellet from 1L was resuspended in 10ml of 0.1M TrisHCl pH 7.2 and stored at -80degC.
Method of Induction	Temperature Shift
Cell Density at Induction	OD 600 = 1.0
Cell Disruption Method	Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	gel filtration
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	0.1M TrisHCl pH 7.2, 2M NaCl
Solubilization Buffer	8M urea, 1mM EDTA, 25mm Tris-PO4, 10mM DTT, 0.2M KCl pH 7.5
Refolding Buffer	50mM Hepes pH 7.5, 20% (w/v) glycerol, 0.2M KCl, 200microM DTT, 100microM Zinc acetate
Pre-Refolding Purification	gel filtration
Tag Cleaved	no tag
Refolding pH	7.5
Refolding Temperature	4.0
Protein Concentration	30microg/ml
Refolding Time	12-16h
Redox Agent	DTT
Redox Agent Concentration	0.2M,0.2M
Refolding Protocol	Frozen cells were thawed and room temperature, then re-pelleted by centrifugation (6000rpm, 15min, 4degC). Each gram of cells was resuspended in 10ml of 0.1 TrisHCl pH 8.0, 10mM EDTA, 1mM DTT and lysozyme was added to 1mg/ml. The cells were incubated on ice for at least one hour, then sonicated. The lysate was then centrifuged (30min, 12500rpm, 4degC). The pellet was then washed twice in ice-cold wash buffer. The washed pellet was subjected to a sucrose gradient, the isolated inclusion bodies from 1L were then resuspended in solubilization buffer and passed down a gel filtration column (TSK4000SW). The selected fractions were pooled and diluted to a final concentration of 30microg/ml with denaturation buffer. The protein was then dialysed for 3h at room temperature aginast 8M urea, 0.2M KCl, 1mM DTT, 1mM Tris-acetate pH 7.0, then dialysed against refolding buffer for 12-16h at 4degC.
Refolding Assay	DNA binding,Bioactivity,Western Blot,SDS-PAGE,Complex Assembly
Refolding Chaperones	None
Refolding Additives	Glycerol
Additives Concentration	20%
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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