Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Myostatin
Abbreviated Name	MSTN
SCOP Family	Transforming growth factor (TGF)-beta
Structure Notes
Organism	Pig (Longissimus dorsi)
UniProt Accession	O18831
SCOP Unique ID	n/a
Structure Solved
Class	Small Proteins
Molecularity	Dimer

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	387
Molecular Weight	44032.2
Pi	6.46749
Molecular Weight	44032.2
Disulphides	3
Full Sequence	MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPTL NENSEQK ENVEKEGLCN ACMWRQNTKS SRLEAIKIQI LSKLRLETAP NISKDAIRQL LPKAPPLREL IDQYDVQRDD SSDGSLEDDD YHATTETIIT MPTESDLLMQ VEGKPKCCFF KFSSKIQYNK VVKAQLWIYL RPVKTPTTVF VQILRLIKPM KDGTRYTGIR SLKLDMNPGT GIWQSIDVKT VLQNWLKQPE SNLGIEIKAL DENGHDLAVT FPGPGEDGLN PFLEVKVTDT PKRSRRDFGL DCDEHSTESR CCRYPLTVDF EAFGWDWIIA PKRYKANYCS GECEFVFLQK YPHTHLVHQA NPRGSAGPCC TPTKMSPINM LYFNGKEQII YGKIPAMVVD RCGCS
Notes	n/a

Expression
Report	Jin HJ, Dunn MA, Borthakur D, Kim YS. (2004) Protein Expression and Purification, 35, 1-10
Project Aim	Functional Studies
Fusion	N-terminal hexahis + pro seq
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21
Expression Temp	37.0
Expression Time	4h
Expression Vector	pCRT7/NT-TOPO
Expression Protocol	200mL prewarmed LB broth containing 100 micrograms/ml ampicillin and 34 micrograms/ml chloramphenicol was inoculated 250 microlitres of overnight cell culture grown at 37degC. Cells were grown until OD600 reached 0.6-0.7 and protein expression was induced with 1mM IPTG and further incubation for 4h, cells were then harvested by centrifugation.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.6-0.7
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	n/a
Solubilization Buffer	50mM CAPS pH 11.0, 0.3% N-laurylsarcosin, 1mM DTT
Refolding Buffer	20mM TrisHCl pH 8.5, 0.15M NaCl, 1mM DTT, 1.3mM reduced glutathione, 1.0mM oxidized glutathione
Pre-Refolding Purification	None
Tag Cleaved	no
Refolding pH	8.5
Refolding Temperature	4.0
Protein Concentration	10 micrograms/ml
Refolding Time	1week
Redox Agent	GSH/GSSG/DTT
Redox Agent Concentration	1.3mM/1mM/1mM,1.3mM/1mM/1mM
Refolding Protocol	Cells were lysed by sonication and 3 cycles of flash freezing in liquid nitrogen and thawing at 37degC. The lysed cells were centrifuged (5000g x 20min), the pellet was wahsed twice with 50mM potassium phosphate, 400mM NaCl, 100mM KCl, 10% glycerol, 0.5% Triton X-100 and 10mM imidazole (pH 7.8). The pellet was then washed a further 2 times with distilled water. The inclusion bodies were solubilized in solubilization buffer for 15min at room temperature and then centrifuged for 10min. The supernatant was then rapidly diluted into cold refolding buffer to a final concentration of 10 micrograms/ml before being vortexed vigourously at 1 min at room temperature. The refoldin gmixter was then incubated at 4degC for one week. Following refolding, the mixture was dialyzed overnight in 20mM TrisHCl (pH 8.5) at 4degC with two buffer changes during dialysis. The refolded dialysed protein was filtered through 0.22 microns. The protein could now be further purified either by anion exchange or affinity chromatography. For anion exchange, 150ml of the protein mixture was loaded onto a Hiprep DEAE equilibrated with 20mM TrisHCl (pH 8.5). After loading, the column was washed with 20mM TrisHCl (pH 8.5) and the protein as then eluted using a linear gradient of 20mM to 1M TrisHCl (pH 8.5) over 60min with a flow rate of 4ml/min. For affinity chromatography, the protein solution (250ml) was loaded onto a nickel effinity column equilibrated with 20mM TrisHCl (pH 8.5), 0.5M NaCl. The column was then washed with equilibration buffer and then step eluted with equilibration buffer containing 20mM and 50mM EDTA. Following anion-exchange or affinity chromatography, the protein was then passed down a Superdex200 size exclusion column. The equilibration and elution buffer for this column was 20mM TrisHCl (pH 8.5), 150mM NaCl.
Refolding Assay	Western Blot
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration
Refolding Yield
Purity
Notes	- similar yield of protein achieved using anion exchange and affinity chromtography - mature processed myostatin (no pro-sequence) also purified by this protocol, but does not refold properly - pro-sequence of unprocessed myostatin removed by furin cleavage after purification (see paper for details

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