Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Troponin T, fast skeletal muscle isoforms
Abbreviated Name	TnT
SCOP Family	Troponin coil-coiled subunits
Structure Notes
Organism	Chicken (Gallus gallus)
UniProt Accession	P12620
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	263
Molecular Weight	31010.7
Pi	6.46
Molecular Weight	31010.7
Disulphides	Unknown
Full Sequence	SDTEEVEHGEEEYEEEAHEAEEVHEEEVHEPAPPPEEAPEEEEKPRIKLTAPKIPEGEKVDFDDIQKKRQNKDLIELQALIDSHFEARRKEEEELVALKERIEKRRAERAEQQRIRAEKEKERQARLAEEKARREEEDAKRKAEDDLKKKKALSSMGASYSSYLAKADQKRGKKQTARETKKKVLAERRKPLNIDHLNEDKLRDKAKELWDWLYQLQTEKYDFAEQIKRKKYEIVTLRNRIDQAQKHSKKAGAKGKVGGRWK
Notes	n/a

Expression
Report	Stone DB, Timmins PA, Schneider DK, Krylova I, Ramos CH, Reinach FC, Mendelson RA. (1998) J Mol Biol, 281, 689-704
Project Aim	Structural Studies
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)pLysS
Expression Temp	0.0
Expression Time	00
Expression Vector	pET3a-TnT
Expression Protocol	CS48/64T-3 (pET3a-TnT; Farah et al., 1994) were transformed into Escherichia coli strain BL21(DE3)pLysS. Protonated troponins were obtained from cells grown on 2 * TY medium. Deuterated troponins were obtained from E. coli cells grown in 95% 2H2O on a hydrolyzate of 98% deuterated Scenedesmus obliquus (Crespi, 1982). Growth in both media was carried out in the presence of ampicillin and chloramphenicol, and expression was induced with IPTG (Studier et al., 1990). Bacteria were harvested by centrifugation and frozen either as a cell pellet or after mixing with an equal volume of 50 mM Tris-HCl (pH 8), 2 mM EDTA.
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Size-exclusion chromatography
Solubility	insoluble

Refolding
Refolding Method	Column refolding: Size-exclusion chromatography
Wash Buffer	8% (w/v) sucrose, 5% (v/v) Triton X-100, 50 mM EDTA, 50 mM sodium citrate (pH 6.5), 1 mM DTT, 0.1 mM PMSF, protease inhibitors
Solubilization Buffer	6 M urea, 50 mM citrate (pH 6.0), 1 mM EDTA, 0.2 mM DTT, 0.1 mM PMSF and protease inhibitorsto
Refolding Buffer	6 M urea, 50 mM citrate (pH 6.0), 1 mM EDTA, 0.2 mM DTT, 0.1 mM PMSF and protease inhibitorsto
Pre-Refolding Purification	Size-exclusion chromatography
Tag Cleaved	no
Refolding pH	6.0
Refolding Temperature	0.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	DTT
Redox Agent Concentration	0.2 mM
Refolding Protocol	TnT was extracted as an insoluble protein (Babbitt et al., 1990) with the pH of the extraction solution set at 6.5, the pI of the TnT-3 isoform (Malnic & Reinach, 1994). Thawed cells were suspended in ten volumes STEC buffer (8% (w/v) sucrose, 5% (v/v) Triton X-100, 50 mM EDTA, 50 mM sodium citrate (pH 6.5), 1 mM DTT, 0.1 mM PMSF, protease inhibitors), sonicated until no longer viscous, and centrifuged at 43,000 g for 30 minutes at 5 C. The pellets were washed once with STEC buffer and dissolved in 6 M urea, 50 mM citrate (pH 6.0), 1 mM EDTA, 0.2 mM DTT, 0.1 mM PMSF and protease inhibitors. Following centrifugation at 245,000 g for 90 minutes at 4 C, the supernatant was loaded directly onto a SP-Sepharose HP column equilibrated in the same solvent and eluted with a 0 to 300 mM NaCl gradient. Fractions containing TnT were pooled, dialyzed against 6 M urea, 50 mM Tris (pH 8), 0.1 mM DTT, protease inhibitors, 0.1 mM PMSF and further purified on Q-Sepharose. The yield was ~10 mg TnT per liter of culture.
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	10 mg/L
Purity	n/a
Notes	Refolding Temperatur is not stated

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