Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Cocaine- and amphetamine-regulated transcript peptide
Abbreviated Name	CART peptide
SCOP Family	Satiety factor CART (cocaine and amphetamine regulated transcript)
Structure Notes
Organism	Rat
UniProt Accession	P49192
SCOP Unique ID	n/a
Structure Solved
Class	Small Proteins
Molecularity	Monomer

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	55
Molecular Weight	6217.2
Pi	8.63021
Molecular Weight	6217.2
Disulphides	3
Full Sequence	IPIYEKKYG QVPMCDAGEQ CAVRKGARIG KLCDCPRGTS CNSFLLKCL E HHHHHH
Notes	n/a

Expression
Report	Couceyro PR and Fritz T (32) Protein Expression and Purification, 32, 185-193
Project Aim	Recombinant Protein Expression
Fusion	C-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	37.0
Expression Time	5h
Expression Vector	pET23b
Expression Protocol	0.5L LB broth containing 34microg/ml chloramphenicol and 100microg/ml ampicillin was inoculated with 0.5ml of an overnight culture. Cells were grown at 37degC until OD600 reached 0.4-0.6. Expression was induced with 1mM IPTG and cells were incubated a further 5h, after which they were harvested by centrifugation (2000g, 10min, 4degC).
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.4-0.6
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Metal affinity chromatography
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	n/a
Solubilization Buffer	6M GdnHCl, 0.5M NaCl, 20mM TrisHCl pH 8.0
Refolding Buffer	50mM TrisHCl pH 8.0, 1mM GSH, 1mM GSSG, 1mM EDTA
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration	<0.1mg/ml
Refolding Time	7 days
Redox Agent	GSH/GSSG
Redox Agent Concentration	1mM/1mM,1mM/1mM
Refolding Protocol	Cells were lysed in solubilization buffer at 5ml buffer/g wet weight of bacterial pellet. Cells were sonicated for 15min on ice and centrifuged (20000g, 30min, 4degC). The supernatant was filtered, then loaded onto a Ni-NTA column with gentle shaking. The resin was washed with 20 column volumes each of solubilization buffer, urea buffer (8M urea, 0.5M NaCl, 20mM TrisHCl pH 8.0), solubilization buffer with 20mM imidazole and urea buffer with 20mM imidazole. The protein was then eluted with solubilization buffer containing 300mM imidazole. The peptide was diluted to 0.5mg/ml and reduced in 150mM DTT, 4M GdnHCl, 50mM TrisHCl pH 8.0 for 4h, then refolded by dilution to less than 0.1mg/ml in refolding buffer and left gently shaking at room temperature for 7 days. Following refolding, the peptide was concentrated on a C18 reverse phase mini-column.
Refolding Assay	Bioactivity,Western Blot,HPLC,SDS-PAGE,Amino acid sequencing,Mass spectrometry
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	95%
Notes	A range of refolding buffers tested (see paper for more details). C-terminal His tag may affect bioactivity of peptide (see paper for more details)

Back to refolding records...