Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Enteropeptidase
Abbreviated Name	Enteropeptidase
SCOP Family	Unknown
Structure Notes
Organism	Human
UniProt Accession	P98073
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Heterodimer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	1035
Molecular Weight	112924.0
Pi	4.9043
Molecular Weight	112924.0
Disulphides	6
Full Sequence	MGSKRGISSRHHSLSSYEIMFAALFAILVVLCAGLIAVSCLTIKESQRGAALGQSHEARA TFKITSGVTYNPNLQDKLSVDFKVLAFDLQQMIDEIFLSSNLKNEYKNSRVLQFENGSII VVFDLFFAQWVSDQNVKEELIQGLEANKSSQLVTFHIDLNSVDILDKLTTTSHLATPGNV SIECLPGSSPCTDALTCIKADLFCDGEVNCPDGSDEDNKMCATVCDGRFLLTGSSGSFQA THYPKPSETSVVCQWIIRVNQGLSIKLSFDDFNTYYTDILDIYEGVGSSKILRASIWETN PGTIRIFSNQVTATFLIESDESDYVGFNATYTAFNSSELNNYEKINCNFEDGFCFWVQDL NDDNEWERIQGSTFSPFTGPNFDHTFGNASGFYISTPTGPGGRQERVGLLSLPLDPTLEP ACLSFWYHMYGENVHKLSINISNDQNMEKTVFQKEGNYGDNWNYGQVTLNETVKFKVAFN AFKNKILSDIALDDISLTYGICNGSLYPEPTLVPTPPPELPTDCGGPFELWEPNTTFSST NFPNSYPNLAFCVWILNAQKGKNIQLHFQEFDLENINDVVEIRDGEEADSLLLAVYTGPG PVKDVFSTTNRMTVLLITNDVLARGGFKANFTTGYHLGIPEPCKADHFQCKNGECVPLVN LCDGHLHCEDGSDEADCVRFFNGTTNNNGLVRFRIQSIWHTACAENWTTQISNDVCQLLG LGSGNSSKPIFSTDGGPFVKLNTAPDGHLILTPSQQCLQDSLIRLQCNHKSCGKKLAAQD ITPKIVGGSNAKEGAWPWVVGLYYGGRLLCGASLVSSDWLVSAAHCVYGRNLEPSKWTAI LGLHMKSNLTSPQTVPRLIDEIVINPHYNRRRKDNDIAMMHLEFKVNYTDYIQPICLPEE NQVFPPGRNCSIAGWGTVVYQGTTANILQEADVPLLSNERCQQQMPEYNITENMICAGYE EGGIDSCQGDSGGPLMCQENNRWFLAGVTSFGYKCALPNRPGVYARVSRFTEWIQSFLH
Notes	n/a

Expression
Report	Gasparian ME, Ostapchenko VG, Schulga AA, Dolgikh DA, Kirpichnikov MP. (2003) Protein Expression and Purification, 31, 133-139
Project Aim	Functional Studies
Fusion	Thioredoxin
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21
Expression Temp	28.0
Expression Time	23h
Expression Vector	pET32a
Expression Protocol	unknown
Method of Induction	Not Stated
Cell Density at Induction	OD =
Cell Disruption Method	French Press
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	partial

Refolding
Refolding Method	Dilution/Dialysis combination
Wash Buffer	unknown
Solubilization Buffer	0.1 M Tris?HCl (pH 8.6), 1 mM EDTA-Na, 20 mM dithiothreitol, and 6 M guanidine?HCl
Refolding Buffer	0.7 M arginine?HCl, 2 mM reduced glutathione, and 1 mM EDTA
Pre-Refolding Purification	None
Tag Cleaved	yes
Refolding pH	8.6
Refolding Temperature	4.0
Protein Concentration
Refolding Time	75h
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Inclusion bodies were sedimented at 50 000g for 20 min, 4 °C. The protein (Trx/L-HEP) was solubilized at room temperature in 10 mL of 0.1 M Tris?HCl (pH 8.6), 1 mM EDTA-Na, 20 mM dithiothreitol, and 6 M guanidine?HCl. L-HEP was refolded by a modification of a protocol for bovine enteropeptidase light chain [24]. Insoluble material was removed by centrifugation at 80 000g for 20 min, 4 °C. The solubilized protein was dialyzed at room temperature against 3 M guanidine?HCl (pH 2.5), mixed with 10 mL of oxidation buffer (50 mM Tris?HCl, pH 8.3, 6 M guanidine?HCl, and 0.1 M oxidized glutathione), and dialyzed against 3 M guanidine?HCl (pH 8.0). To initiate disulfide exchange and refolding, the protein solution was dropwise diluted in 600 mL of 0.7 M arginine?HCl (pH 8.6), 2 mM reduced glutathione, and 1 mM EDTA, and incubated for 75 h at 4 °C. After dialyzing the reaction mixture for 2 h against 0.1 M Tris?HCl (pH 8.0) containing 1 M CaCl2 at room temperature, complete autocatalytic cleavage of fusion protein took place.
Refolding Assay	Enzyme activity
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	NULL
Refolding Yield	2%
Purity	few very faint bands on SDS PAGE
Notes

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