Refolding Record:

Protein See all refolding records for this protein...
Protein Name	APG8a (At4g21980) autophagy protein
Abbreviated Name	APG8a
SCOP Family	Unknown
Structure Notes
Organism	Arabidopsis thaliana
UniProt Accession	Q8LEM4
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Monomer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	155
Molecular Weight	17184.7
Pi	9.35065
Molecular Weight	17184.7
Disulphides	0
Full Sequence	MGSSHHHHHHSSGLVPRGSH MAKSSFKISN PLEARMSESS RIREKYPDRI PVIVEKAGQS DVPDIDKKKY LVPADLTVGQ FVYVVRKRIK LGAEKAIFVF VKNTLPPTAA LMSAIYEEHK DEDGFLYMTY SGENTFGSLT VA RAPPPPPPLRSGC
Notes	n/a

Expression
Report	Chae YK, Im H, Zhao Q, Doelling JH, Vierstra RD, Markley JL. (2004) Protein Expression and Purification, 34, 280-283
Project Aim	NMR
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	Rosetta(DE3)
Expression Temp	37.0
Expression Time	3h
Expression Vector	pET28a
Expression Protocol	An 10mL overnight culture of cells grown in LB media (50 micrograms/ml kanamycin, 34 micrograms/ml chloramphenicol) was used to inoculate 1L LB media (containing same antibiotics). Cells were grown until OD600 reached 1.0, protein expression was induced by the addition of 1mM IPTG and grown for a further 3h. Cells were harvested by centrifugation and resuspended in 50ml of 10mM TrisHCl pH 8.0.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 1.0
Cell Disruption Method	Freeze-thaw
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	partial

Refolding
Refolding Method	Column refolding: Nickel-chelating chromatography
Wash Buffer	20mM sodium phosphate pH 7.0, 300mM NaCl, 10mM imidazole, urea (8,4,2,1M)
Solubilization Buffer	8M GdnHCl, 10mM TrisHCl, pH 8.0
Refolding Buffer	20mM sodium phosphate pH 7.0, 300mM NaCl, 10mM imidazole
Pre-Refolding Purification	None
Tag Cleaved	yes
Refolding pH	7.0
Refolding Temperature	25.0
Protein Concentration
Refolding Time
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Cells were lysed by thawing with 3mg of DNaseI. GdnHCl was added to lysed cells to a final concentration of 8M, the denatured cell lysate was then centrifuged (20min x 26000g). The supernatant was loaded onto a Ni-agarose column, which was washed with 50ml aliquots of wash buffer with sequentially decreasing concentrations of urea (8,4,2,1,0M). The refolded protein was eluted with 20mM sodium phosphate pH 7.0, 300mM NaCl, 0.5M urea, 1% glycerol, 300mM imidazole. Imidazole concentration was reduced by ultrafiltration. The N-terminal his-tag was removed by incubation with biotinylated thrombin at 4degC for 18h. The reaction mixture was then passed down the Ni-agarose to remove the His-tag, and the thrombin was reomved by batch-mode chrompatography using avidin-agarose resin slurry.
Refolding Assay	15N -1H chemical shifts (ppm)
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration
Refolding Yield
Purity
Notes	Urea and glycerol included in final buffer to minimize intermolecular interactions (and hence aggregation). Relatively high final NaCl concentration reduces intermolecular ionic interactions.

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