Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Parathyroid hormone/parathyroid hormone-related peptide receptor
Abbreviated Name	PTHR1
SCOP Family	Parathyroid hormone fragments (residues between 1 and 39)
Structure Notes
Organism	Human
UniProt Accession	Q03431
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Monomer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	167
Molecular Weight	19169.6
Pi	6.25
Molecular Weight	19169.6
Disulphides	3
Full Sequence	ALVDADDVMTKEEQIFLLHRAQAQCEKRLKEVLQRPASIMESDKGWTSASTSGKPRKDKASGKLYPESEEDKEAPTGSRYRGRPCLPEWDHILCWPLGAPGEVVAVPCPDYIYDFNHKGHAYRRCDRNGSWELVPGHNRTWANYSECVKFLTNETREREVFDRLGMI
Notes	n/a

Expression
Report	Grauschopf U, Lilie H, Honold K, Wozny M, Reusch D, Esswein A, Schäfer W, Rücknagel KP, Rudolph R. (2000) Biochemistry, 39, 8878-8887
Project Aim	Expression and charactrization
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)
Expression Temp	37.0
Expression Time	3 h
Expression Vector	pET15b
Expression Protocol	For expression of nPTHR, the plasmid was transferred into the E. coli strain BL21(DE3). Since translation of eucaryotic transcription products in E. coli is often impaired by differences in codon usage, especially by the rare codons for L-arginine, AGA and AGG, we cotransfected the host cells with an additional plasmid, pUBS520, that enables constitutive expression of the corresponding tRNA (17, 18). For shake flask growth, 1 L LB-medium supplemented with kanamycin (25 µg/mL) and ampicillin (100 µg/mL) was inoculated with 10 mL overnight culture and cultivation was performed at 37 °C. At an optical density of OD546 nm ) 0.8, protein expression was induced with 1 mM IPTG. After an additional 3 h incubation, cells were harvested by centrifugation (15 min; 4000g).
Method of Induction	IPTG
Cell Density at Induction	OD 0.8 = 546
Cell Disruption Method	high-pressure dispersion
Lytic Agent	None
Pre-Refolding Purification	Metal affinity chromatography
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	0.1 M TRIS, 150 mM NaCl, pH 7
Solubilization Buffer	0.1 M TRIS, 6 M guanidinium chloride, 100 mM DTT, and 1 mM EDTA, pH 8
Refolding Buffer	1 M L-arginine, 0.05 M sodium phosphate, 1 mM EDTA, 5 mM GSH, and 1 mM GSSG, pH 8
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	yes
Refolding pH	8.0
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	4 days
Redox Agent	GSH/GSSG
Redox Agent Concentration	5/1 mM
Refolding Protocol	The obtained pellet was resuspended in 0.1 M TRIS, 150 mM NaCl, pH 7, containing 0.5% LDAO. Followed by a second washing step, the washing procedure was repeated twice without LDAO. The collected inclusion bodies were stored at - 20 °C until use. After inclusion bodies were solubilized in 0.1 M TRIS, 6 M guanidinium chloride, 100 mM DTT, and 1 mM EDTA, pH 8 (10 mg of inclusion body material/mL), at room temperature for 2 h, insoluble material was removed by centrifugation (30 min; 30000g). To remove DTT, the solubilized protein was extensively dialyzed against 4 M guanidinium chloride and 0.1 M sodium phosphate, pH 6. To obtain binding of the His-tagged protein to an IMAC column (Qiagen), the pH of the solution was readjusted to pH 8 by adding 1 M NaOH. After washing the column with 0.1 M sodium phosphate and 4 M guanidinium chloride, pH 6.3, nPTHR was eluted by a pH shift to pH 4.5. Renaturation was achieved by dialysis against 15 vol of renaturation buffer (1 M L-arginine, 0.05 M sodium phosphate, 1 mM EDTA, 5 mM GSH, and 1 mM GSSG, pH 8) at a protein concentration of 0.5-1 mg/mL for 4 days at 4 °C.
Refolding Assay	Analytical centrifugation
Refolding Chaperones	None
Refolding Additives	L-Arginine
Additives Concentration	1 M
Refolding Yield	30-50%
Purity	n/a
Notes	n/a

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