Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Nikkomycin biosynthesis protein SanV
Abbreviated Name	sanV
SCOP Family	Unknown
Structure Notes
Organism	Streptomyces ansochromogenes
UniProt Accession	Q83WU0
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	423
Molecular Weight	45750.9
Pi	5.97
Molecular Weight	45750.9
Disulphides	Unknown
Full Sequence	MAPPTGPGRFTRFVRRASSEGKLVVQPRMGFGTVEQMRAGLDAVRTVDAATVGTITVDSYTRVNDHASALLALEKGADLNGFPLVAHGAAVTRELLAGIAGDDFPVQVRHGSALPGALFEGLVAAGVDATEGGPVSYCLPYSRVPLAQAVDAWAECCEMLAGISEPVHLESFGGCMLGQLCPPSLLISLSILEGLFFREHGVRDISVSYAQQTNQQQDIEAIHALRGLAKEWLGDTDWHAVLYTYMGVYPRTRQGAYGLLEESARLAARSGTERLIVKTAVEASRIPSITENVDALERAAWAAEREAVAEPAGIPDSGIYEEAQAIITHTLTLGSDVGKALVRAFALGHLDIPFCLHQDNANRCRARIDDRGRLTWADPAGVPIPRARDLARNRQRLTARGLIDMLSYNERRYDHPSRTSHPR
Notes	n/a

Expression
Report	Li Y, Ling H, Li W, Tan H (2005) Metabolic Engineering, 7, 165-173
Project Aim	Drug Studies
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	37.0
Expression Time	4 h
Expression Vector	pET23b
Expression Protocol	The PCR products of sanU and sanV were purified and digested with NdeI and HindIII, then ligated into the same sites of pET23b to generate pET23b::sanU and pET23b::sanV, respectively. Plasmids pET23b::sanU and pET23b::sanV were designed to give C-terminally His-tagged SanU and SanV, respectively. They were introduced into E. coli BL21 (DE3) for overexpression.The resulting transformants containing pET23b::sanU were grown at 37 °C overnight in LB medium with ampicillin (100 μg/ml). 0.5 ml of the above overnight culture was inoculated to 100 ml LB medium and incubated at 37 °C on a rotary shaker. At OD600=0.4, the culture was induced by 0.5 mM IPTG and grown at 25 °C for a further 4 h before harvesting.
Method of Induction	IPTG
Cell Density at Induction	OD 0.4 = 600
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Metal affinity chromatography
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	100 mM PBS buffe
Solubilization Buffer	6 M guanidine hydrochloride
Refolding Buffer	0.1 M potassium phosphate buffer, pH 7.6, containing 0.5 M guanidine hydrochloride, 10% (v/v) glycerol and 1 mM DTT
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no tag
Refolding pH	7.6
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	DTT
Redox Agent Concentration	1 mM
Refolding Protocol	The supernant was removed and the pellet was collected, and solubilized in binding buffer with 6 M guanidine hydrochloride. The solution was stirred at room temperature for 1 h and then insoluble material was removed by centrifugation at 13000 rpm for 15 min. Subsequently, solubilized SanV was applied to Ni-NTA affinity chromatography column according to the procedure of purification for inclusion body. The elution containing solubilized SanV was dialyzed in 100 mM PBS buffer to remove guanidine hydrochloride. Then, solubilized SanV was refolded according to method mentioned by Holloway et al. (1996), except that 0.5 M instead of 1 M guanidine hydrochloride was used. During the refolded process, the solution was stirred gently for 1 h and the residual guanidine hydrochloride was removed by dialysis against 100 mM PBS buffer containing 1 mM DTT and 10% (v/v) glycerol. The dialytic sample was cleared by centrifugation at 13000 rpm for 10 min and then concentrated to 4 mg/ml.
Refolding Assay	enzyme activity
Refolding Chaperones	None
Refolding Additives	Glycerol
Additives Concentration	10%
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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