Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Thrombopoietin receptor
Abbreviated Name	Thrombopoietin receptor
SCOP Family	Unknown
Structure Notes
Organism	Human
UniProt Accession	P40238
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	516
Molecular Weight	57906.7
Pi	5.66651
Molecular Weight	57906.7
Disulphides	4
Full Sequence	MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSD QDVSL LASDSEPLKC FSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTR YVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIK AMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIAT ETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGS CLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQ CFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKT NPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVR LPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEP PLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETA
Notes	n/a

Expression
Report	Ng CKF, Huxtable S, Xu P, Hsieh DPH (2001) Protein Expression and Purification, 21, 129-133
Project Aim	Biophysical Studies
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)pLysS
Expression Temp	30.0
Expression Time	3h
Expression Vector	pET30a
Expression Protocol	1L culture of cells was grown in LB medium with 30microg/ml kanamycin at 37degC until A650nm reached 0.6. Protein expression was induced with 1mM IPTG, then the culture was incubated at 30degC for a further 3h. The bacteria were harvested by centrifugation (4420g, 15min, 4degC) and the pellet was stored at -20degC.
Method of Induction	IPTG
Cell Density at Induction	OD 650 = 0.6
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Metal affinity chromatography
Solubility	insoluble

Refolding
Refolding Method	Column refolding: Size-exclusion chromatography
Wash Buffer	5mM Imidazole, 0.5M NaCl, 20mM TrisHCl pH 7.9
Solubilization Buffer	5mM Imidazole, 0.5M NaCl, 20mM TrisHCl pH 7.9, 8M urea
Refolding Buffer	20mM NaHPO4, 200mM NaCl, 1mM DTT, pH 7.4
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no
Refolding pH	7.4
Refolding Temperature	25.0
Protein Concentration
Refolding Time
Redox Agent	DTT
Redox Agent Concentration	1mM
Refolding Protocol	The cell pellet was resuspended with 100ml of 50mM TrisHCl pH 8.0 with 2mM EDTA and 1-ml 1% Triton X-100. The mixture was chilled on ice for 30min and lysed by sonication. The lysate was centrifuged (4420g, 15min, 4degC) and the isolated inclusion bodies were then further purified by repeated washing and differential centrifugation in wash buffer. The purified inclusion bodies were then resuspended in 100ml solubilization buffer for 2h at 4degC. 2-mercaptoethanol was added to a final concentration of 10mM and the mixture was then kept at 4degC for at least 1h. The solubilized inclusion bodies were centrifuged (12000g, 1min) and the supernatant was applied to a metal chelated column charged with 2.5ml of 0.1M CuSO4 and pre-equilibrated with solublization buffer. The column was washed with 20ml solubilization buffer and then the bound protein was eluted with a step imidazole gradient using different proportions of solubilization buffer mixed with lution buffer (8M urea, 1M imidazole, 0.5M NaCl, 20mM TrisHCl, pH 7.9) at a flow rate of 5ml/min. 1ml fractions were collected. Selected fractions were pooled then refolded by passage through a Superdex 75 HR 10/30 gel filtration column preequilibrated with refolding buffer at a flow rate of 0.5ml/min. 1ml fractions were collected
Refolding Assay	Surface plasmon resonance binding
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration
Refolding Yield	3mg/L culture
Purity	80-85%
Notes

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