Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Photoactivated adenylate cyclase subunit alpha
Abbreviated Name	PAC alpha
SCOP Family	Unknown
Structure Notes
Organism	Euglena gracilis
UniProt Accession	Q8S9F2
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Unknown

Construct
Full Length	n
Domain	BLUF 2
Chimera	n/a
Variants	n/a
Chain Length	93
Molecular Weight	10295.0
Pi	9.16
Molecular Weight	10295.0
Disulphides	Unknown
Full Sequence	LITLTYISQAAHPMSRLDLASIQRIAFARNESSNITGSLLYVSGLFVQTLEGPKGAVVSLYLKIRQDKRHKDVVAVFMAPIDERVYGSPLDMT
Notes	n/a

Expression
Report	Ito S, Murakami A, Sato K, Nishina Y, Shiga K, Takahashi T, Higashi S, Iseki M, Watanabe M. (2005) Photochem Photobiol Sci, 4, 762-769
Project Aim	Expression and identification
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	6 h
Expression Vector	pET21d(28a)-F2
Expression Protocol	The recombinant plasmids pET21d(28a)-F2 and were introduced into E. coli strain BL21(DE3) (EMD Biosciences) and cultured as described above. The cells were cultured in LB containing 100 lg ml−1 ampicillin. 6His-F2 and was overexpressed by induction with 0.5 mM IPTG at 37 ◦ C for 6 h. Almost all of the expressed 6His-F2 and was present in insoluble inclusion bodies. Harvested cells were suspended in potassium phosphate buffer, pH 7. The cells were broken by ultrasonication. Inclusion bodies were separated from the sonicate by centrifugation at 10 000 g for 30 min at 4 ◦ C
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	ultrasonication
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	50 mM potassium phosphate buffer, pH 7, containing 4% Triton X-100,
Solubilization Buffer	8 M guanidine-HCl311
Refolding Buffer	50 mM potassium phosphate buffer, pH 7, containing 20 uM exogenous flavins, FAD (F-6625, Sigma, St. Louis, MO, USA) or flavin mononucleotide (FMN) F-8399, Sigma)
Pre-Refolding Purification	None
Tag Cleaved	no
Refolding pH	7.0
Refolding Temperature	24.0
Protein Concentration	n/a
Refolding Time	20 min
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	pellet was resuspended and washed with 50 mM potassium phosphate buffer, pH 7, containing 4% Triton X-100, and finally suspended in 50 mM potassium phosphate buffer, pH 7. An aliquot of the inclusion body suspension, containing 4.2 mg ml−1 total protein for 6His-F2, was mixed with 3 parts of 8 M guanidine-HCl solution, resulting in a denatured protein solution. Protein-refolding of 6His-F2 was performed by rapid manual mixing of an aliquot of the suspension of denatured 6His-F2 solution with 12.5 parts of the refolding buffer: 50 mM potassium phosphate buffer, pH 7, containing 20 lM exogenous flavins, FAD (F-6625, Sigma, St. Louis, MO, USA) or flavin mononucleotide (FMN) (F-8399, Sigma). The assembling efficiencies for 6His-F2 were low when refolding buffers with pH lower than 7 were used. Protein-refolding of 6His-F1 was performed using the refolding buffer containing 20 lM FAD. The refoldings of denatured 6His-F1 and 6His-F2 were completed by stabilization at ca. 24 ◦ C for 20 min in darkness. After the insoluble proteins were removed from the refolded protein solutions by centrifugation at 10 000 g for 30 min at 4 ◦ C, purification of 6His-F1 and 6His-F2 was performed using His-tag-binding resin (Talon metal affinity resin, BD Biosciences Clontech, Palo Alto, CA, USA) and elution buffer as described by the manufacturer.
Refolding Assay	Biochemical analyses,Spectrophotometric analyses
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

Back to refolding records...