Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Cyclophilin also known as Peptidyl-prolyl cis-trans isomerase A
Abbreviated Name	CypA
SCOP Family	Cyclophilin (peptidylprolyl isomerase)
Structure Notes
Organism	Human
UniProt Accession	P62937
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	165
Molecular Weight	17881.3
Pi	7.82
Molecular Weight	17881.3
Disulphides	Unknown
Full Sequence	VNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFGYKGSCFHRIIPGFMCQGGDFTRHNGTGGKSIYGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFICTAKTEWLDGKHVVFGKVKEGMNIVEAMERFGSRNGKTSKKITIADCGQLE
Notes	n/a

Expression
Report	Xu LR, Yan X, Luo M, Guan YX, Yao SJ. (2008) J Biol Chem, 24, 302-310
Project Aim	Over expression & Renaturation
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)
Expression Temp	30.0
Expression Time	9 h
Expression Vector	pAVD30
Expression Protocol	Preparation of Recombinant CypA. The pAVD30 plasmid encoding CypA was transformed into E. coli BL21(DE3) strain by the calcium chloride method and transformants were screened on 2×YT plates containing 150 g/mL ampicillin. Cells were grown aerobically in the medium composed of tryptone 16 g/L, yeast extract 10 g/L, NaCl 5 g/L, and mannitol 10 g/L, which was supplemented with 20 mg/L ampicillin. The transformant was precultured overnight under 37 C, 220 rpm, and inoculated to 200 mL medium in a 500 mL shake flask with a volume ratio of 10%. When the OD600 reached 1.3, IPTG was added to the final concentration of 1 mM to induce the expression of CypA, and the cultivation condition was adjusted to 30 C and 180 rpm. After incubation for additional 9 h, cells were harvested by centrifugation at 8 000 rpm for 10 min at 4 C. The wet cells were washed with 50 mM Tris-HCl (pH 8.5), and resuspended in 10% volume of the fermentation broth of the same buffer. The cells were lysed by ultrasonication. After discarding of the cell debris by centrifugation at 14 000 rpm for 40 min at 4 C, 6 mL of supernatant was loaded onto ion exchange chromatography where a 25 mL DEAE Sepharose Fast Flow resin column was used. The purification process was carried on an ÄKTA Explorer 100 (GE healthcare, USA) and the absorbance at 280 nm was monitored. Purified CypA fractions can be collected and protein concentration was determined by denaturing SDS-PAGE and Bradford method.
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	ultrasonication
Lytic Agent	None
Pre-Refolding Purification	DEAE-Cellulofine column chromatography
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	50 mM Tris-HCl (pH 8.5)
Solubilization Buffer	8 M urea and 10 mM DTT
Refolding Buffer	100 mM K2HPO4/KH2PO4 (pH 7.0)
Pre-Refolding Purification	DEAE-Cellulofine column chromatography
Tag Cleaved	no tag
Refolding pH	7.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Refolding of CypA Protein. Different fractions of 100 M CypA denatured in 8 M urea and 10 mM DTT were diluted to 100 mM K2HPO4/KH2PO4 (pH 7.0) buffer to initiate refolding (23). Refolding temperature was 25 C. The refolding kinetics was monitored by emission at 350 nm (5 nm bandwidth) after excitation at 280 nm (2.5 nm bandwidth). Refolded CypA was further tested for the recovery of PPIase activity. The aggregate in a concentrated initially 10 M refolding sample was investigated using native PAGE.
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

Back to refolding records...