Refolding Record:

Protein See all refolding records for this protein...
Protein Name	5-hydroxytryptamine 4 receptor
Abbreviated Name	5-HT-4
SCOP Family	Unknown
Structure Notes
Organism	Mouse
UniProt Accession	P97288
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Monomer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	395
Molecular Weight	44883.8
Pi	8.38
Molecular Weight	44883.8
Disulphides	1
Full Sequence	MDKLDANVSSNEGFRSVEKVVLLTFLAVVILMAILGNLLVMVAVCRDRQLRKIKTNYFIV SLAFADLLVSVLVMPFGAIELVQDIWAYGEMFCLVRTSLDVLLTTASIFHLCCISLDRYY AICCQPLVYRNKMTPLRIALMLGGCWVLPMFISFLPIMQGWNNIGIVDVIEKRKFSHNSN STWCVFMVNKPYAITCSVVAFYIPFLLMVLAYYRIYVTAKEHAQQIQMLQRAGATSESRP QPADQHSTHRMRTETKAAKTLCVIMGCFCFCWAPFFVTNIVDPFIDYTVPEQVWTAFLWL GYINSGLNPFLYAFLNKSFRRAFLIILCCDDERYKRPPILGQTVPCSTTTINGSTHVLRD AVECGGQWESRCHLTATSPLVAAQPSDTEHHHHHH
Notes	n/a

Expression
Report	Baneres, J.L., Mesnier, D., Martin, A., Joubert, L., Dumuis, A., Bockaert, J. (2005) J Biol Chem, 280, 20253-20260
Project Aim	Drug Studies
Fusion	N-terminal KSI sequence, C-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	Rosetta(DE3)pLysS
Expression Temp	37.0
Expression Time	4h
Expression Vector	pET31b
Expression Protocol	Transformed cells grown in 2YT medium containing 100mg/L and then at the same time cells induced 0.2
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 1
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Size-exclusion chromatography
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	1M urea, 25mM TrisHCl, pH 7.8, 500mM NaCL, 10microg/ml leupeptin, 10 microg/ml aprotinin and 0.5 mM phenylmethylsulfonyl fluoride
Solubilization Buffer	25 mM TrisHCl, pH 7.8, 6 M urea, 0.2 (w/w) SDS, 0.2 2-mercaptoethanol
Refolding Buffer	12.5 sodium borate, 25mM NaCl, 2mM MgCl2, containing 1% DMPC, 1% CHAPS, 0.02 cholesteryl hemisuccinate
Pre-Refolding Purification	Size-exclusion chromatography
Tag Cleaved	no
Refolding pH	9.2
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	Beta-mercaptoethanol
Redox Agent Concentration	0.2,0.2
Refolding Protocol	Inclusion bodies were washed x2 in washing buffer, and solubilized. The solubilized proteins were loaded onto a Ni2+-NTA-agarose column. After washing with solubilization buffer, a linear imidazol gradient (0-200mM) in solubilization buffer was used to elute protein. The eluted fractions were then analysed by SDS page, and dialyzed against refolding buffer. Ketoisomerase fusion partner was removed via acid or cleavage with factor XA. To do this fusion protein was dialyzed in 50 mM TrisCl, 50mM NaCL, 1 mM CaCl2, 5mM lauryldimethylamino oxide, pH 6.5 and digested for 3hr at 20degC. Reaction stopped by addition of PMSF to a final concentration of 1 mM and reaction was dialysed against solubilisation buffer. Urea denatured 5HT4 was loaded on a Ni-NTA matrix and washed with buffer(12.5 sodium borate, 10mM KCL, 2mM MgCl2, 6 M urea, 1.25% (w/v) sarcosyl, pH 8.0). Refolding was achieved by applying linear gradient from buffer above buffer to: (12.5 sodium borate, 25mM NaCl, 2mM MgCl2, containing 1% DMPC, 1% CHAPS, 0.02 cholesteryl hemisuccinate) Dissociation from matrix occurred via washing with refolding buffer containing imidazole. Dissociated proteins were then dialyzed against 12.mM sodium borate, 25 mM KCL, pH 7.8 containing 1% DMPC, 1 % CHAPS and 0.2% cholesteryl hemisuccinate.
Refolding Assay	Far-UV Circular Dichroism
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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