Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Trypsinogen
Abbreviated Name	Trypsinogen
SCOP Family	Eukaryotic Proteases
Structure Notes
Organism	Human
UniProt Accession	P07477
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Monomer

Construct
Full Length	n
Domain	n/a
Chimera	N terminal fusion protein: first 11 amino acids of protein 10 (major coat protein) of phage T7
Variants	n/a
Chain Length	246
Molecular Weight	26398.7
Pi	6.41355
Molecular Weight	26398.7
Disulphides	5
Full Sequence	MAS MTGGQQMGRGS APFDD DDKIVGGYNC EENSVPYQVS LNSGYHFCGG SLINEQWVVS AGHCYKSRIQ VRLGEHNIEV LEGNEQFINA AKIIRHPQYD RKTLNNDIML IKLSSRAVIN ARVSTISLPT APPATGTKCL ISGWGNTASS GADYPDELQC LDAPVLSQAK CEASYPGKIT SNMFCVGFLE GGKDSCQGDS GGPVVCNGQL QGVVSWGDGC AQKNKPGVYT KVYNYVKWIK NTIAANS
Notes	n/a

Expression
Report	Hohenblum H, Vorauer-Uhl K, Katinger H, Mattanovich D (2004) J Biotechnology, 109, 3-11
Project Aim	Undefined
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	K12 HMS174(DE3)
Expression Temp	37.0
Expression Time	4 h
Expression Vector	pET3a
Expression Protocol	E.coli cells were grown in baffled shake flasks in M9LB medium (per litre: 10g bactotryptone, 5g yeast extract, 5g NaCl, 1g NH4Cl, 3g KH2PO4, 6g Na2HPO4, 4g glucose, 0.25g MgSO4.7H2O) at 37degC. When OD600 was 1.0, cells were induced with 0.4mg/ml IPTG and incubated for a further 4h.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 1.0
Cell Disruption Method	Chemical
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Diafiltration
Wash Buffer	8M urea, 100mM TrisHCl pH 8.6, 1mM EDTA
Solubilization Buffer	9M urea, 100mM TrisHCl pH 8.6, 1mM EDTA, 10mM DTE
Refolding Buffer	50mM TrisHCl pH 8.6, 50mM CaCl2, 3mM reduced glutathione, 0.3mM GSSG
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	8.6
Refolding Temperature	5.0
Protein Concentration
Refolding Time	3 h
Redox Agent	GSH/GSSG
Redox Agent Concentration	3mM/0.3mM
Refolding Protocol	Following centrifugation, cells were lysed with Triton X-100 (0.4% in 10mM TrisHCl pH 8.2, 15mM MgCl2). The insoluble fraction was centrifuged and washed twice in 3-4M Urea. The pellet was resupended in solubilization buffer and incubated for 2.5h at 37degC. To remove DTE, the buffer of the supernatant was changed by ultradiafiltration under nitrogen with washing buffer using an Amicon YM10 membrane and an Amicon stirring chamber(Millipore). A 10-fold dilution of DTE was sufficiant to allow for the formation of mixed disulfides. Then an equal volume of 300mM oxidized glutathion in 8M urea was added and the mixture incubated for 3h. Finally, the buffer was changed again by ultradiafiltration with washing buffer. The optimum dilution of GSSG was 30-fold. The protocol for refolding was optimized based on the dilution method. The best conditions were as follows; Refolding buffer: 50mM TrisHCl pH 8.6, 50mM CaCl2, 3mM reduced glutathion, 0.3mM GSSG. The final trypsinogen concentration was 10 micrograms per ml, the final urea concentration owas 0.4M. Incubation took place at 5degC for 3 hours under nitrogen atmosphere. IN order to increase the trypsinogen concentration in the refolding reaction, a continuous dilution reactor based on cross flow ultradiafiltration was used (see published report for details). Diafiltration was performed over an Amicon Miniplate Bioconcentrator with a YM10 membrane (Millipore)
Refolding Assay	Bioactivity
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	NULL
Refolding Yield
Purity
Notes

Back to refolding records...