Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Fimbrial subunit also known as pilin subunit
Abbreviated Name	n/a
SCOP Family	Unknown
Structure Notes
Organism	Xenorhabdus nematophila
UniProt Accession	Q8GE68
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	179
Molecular Weight	18873.6
Pi	6.4
Molecular Weight	18873.6
Disulphides	Unknown
Full Sequence	MKLNTIGDGFRFWCGFNPGAANAAPTQGDGAVKFTGSIINAACSIKNNNIEVNMGQINNVVLKDGGESKPEHFKIELKDCVLDTLKGVKTMFTGPEAEGTMPGLLALDGGAQGAAIMITNHGNKHIPLGQASDMMTLHDGSKDLNFSARLKGLKGTEGKDISVKTGEFTAIANFTLNYL
Notes	n/a

Expression
Report	Khandelwal P, Choudhury D, Birah A, Reddy MK, Gupta GP, Banerjee N. (2004) Journal of Bacteriology, 186, 6465-6476
Project Aim	Undefined
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)
Expression Temp	37.0
Expression Time	4 h
Expression Vector	pET23a
Expression Protocol	For expression and purification of the recombinant 17-kDa protein from PK4, this strain was grown at 37°C in LB medium with 100 μg of ampicillin per ml and 25 μg of kanamycin per ml. After induction with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 4 h, cells were harvested by centrifugation at 6,000 × g for 10 min at 4°C
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Ni-NTA column
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	1% deoxycholic acid and several times with 10 mM sodium phosphate buffer (pH 8.0)
Solubilization Buffer	8 M urea in 100 mM sodium phosphate buffer-10 mM Tris-Cl [pH 8.0]
Refolding Buffer	100 mM sodium phosphate, 10 mM Tris-Cl; pH 8.0
Pre-Refolding Purification	Ni-NTA column
Tag Cleaved	yes
Refolding pH	8.0
Refolding Temperature	0.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	The pellet was washed three times with 1% deoxycholic acid and several times with 10 mM sodium phosphate buffer (pH 8.0). Then the pellet was suspended in 5 ml of denaturing buffer (8 M urea in 100 mM sodium phosphate buffer-10 mM Tris-Cl [pH 8.0]) and dialyzed against 10 mM sodium phosphate buffer (pH 8.0). For purification, the dialyzed protein was loaded onto a DEAE ion-exchange column and washed with several column volumes of wash buffer (10 mM sodium phosphate, pH 8.0), and the proteins were eluted with an NaCl gradient (0 to 250 mM) in wash buffer. Fractions containing the recombinant protein were pooled and dialyzed against the wash buffer or 10 mM Tris-Cl (pH 8.0).All the other strains were grown at 37°C in LB medium with 100 μg of ampicillin per ml. Expression was induced with 1 mM IPTG for 4 h at 37°C, and the cells were harvested by centrifugation at 6,000 × g for 10 min at 4°C. Each cell pellet was resuspended in 5 ml of denaturing buffer and incubated at 37°C for 1 h on a rotary shaker. The lysate was centrifuged at 12,000 × g for 45 min at room temperature, and the supernatant was loaded onto an Ni-nitrilotriacetic acid (NTA) column previously equilibrated with the denaturing buffer. Recombinant protein bound to Ni-NTA was refolded by using a 8 to 0 M urea gradient in renaturation buffer (100 mM sodium phosphate, 10 mM Tris-Cl; pH 8.0). The proteins were eluted in the renaturation buffer with 250 mM imidazole. Fractions containing the recombinant protein were pooled and dialyzed against 10 mM Tris-Cl or 10 mM sodium phosphate (pH 8.0).
Refolding Assay	Far-UV Circular Dichroism
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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