Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Glutamate receptor 2
Abbreviated Name	GluR2
SCOP Family	Phosphate binding protein-like
Structure Notes
Organism	Rat
UniProt Accession	P19491
SCOP Unique ID	n/a
Structure Solved
Class	Alpha/Beta
Molecularity	Monomer

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	290
Molecular Weight	32222.8
Pi	8.46
Molecular Weight	32222.8
Disulphides	0
Full Sequence	M HHHHHHHH SSGLVPR SGNDTSG LENKTVVVTT ILESPYVMMK KNHEMLEGNE RYEGYCVDLA AEIAKHCGFK YKLTIVGDGK YGARDADTKI WNGMVGELVY GKADIAIAPL TITLVREEVI DFSKPFMSLG ISIMIKKP GTDGN PIESAEDL SKQTEIAYGT LDSGSTKEFF RRSKIAVFDK MWTYMRSAEP SVFVRTTAEG VARVRKSKGK YAYLLESTMN EYIEQRKPCD TMKVGGNLDS KGYGIATPKG SSLGNAVNLA VLKLNEQGLL DKLKNKWWYD KGECGS
Notes	Domains S1 and S2 joined by a 5-amino acid linker (GTDGN)

Expression
Report	Chen G, Su Y, Jin R, Gouaux E (1998) Protein Science, 7, 2623-2630
Project Aim	Crystallography
Fusion	N-terminal poly-histidine 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	2h
Expression Vector	pETGQ
Expression Protocol	Cells were grown at 37degC for 3h, expression was then induced with 1mM IPTG and cells were incubated for an additional 2h. Cells were harvested by centrifugation (4000rpm, 20min)
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	French Press
Lytic Agent	Lysozyme
Pre-Refolding Purification	Size-exclusion chromatography
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	1:50mM TrisHCl pH 8.0, 10mM EDTA, 100mM NaCl, 0.5% Triton X-100, 1mM PMSF, 2: 20mM TrisHCl pH 7.4, 1mM EDTA, 200mM NaCl, 1mM PMSF
Solubilization Buffer	50mM TrisHCl pH 7.4, 5mM EDTA, 8M GdnHCl, 50mM DTT
Refolding Buffer	10mM NaCl, 0.4mM KCl, 0.65M arginine-HCl, 1mM EDTA, pH 8.5
Pre-Refolding Purification	Size-exclusion chromatography
Tag Cleaved	no
Refolding pH	8.5
Refolding Temperature	4.0
Protein Concentration	1-1.5mg/ml
Refolding Time	overnig
Redox Agent	GSH/GSSG
Redox Agent Concentration	1mM/0.2mM,1mM/0.2mM
Refolding Protocol	Cells were harvested, then resuspended in 100ml of Buffer 5 with with 100mg lysozyme and 120mg deoxycholate. 1mg DNase I and 100microL of 1M PMSF. Cells wer disrupted by French press, and the suspension was then centrifuged (17000rpm, 30min). The inclusion bodies were washed with 200ml wash buffer 1, followed by wash buffer 2 with. The washed inclusion bodies from 10L culture were dissolved in 90ml solubilization buffer at room temperature for 4h and the solution was centrifuged (40000rpm, 20degC, 1h) and the supernatant was diluted with 500ml Buffer 8 (20mM NaOAc, 4M GdnHCl, 1mM EDTA, 1mM DTT, pH 4.5). The diluted solution was stirred for 1h at 4deg and centrifuged (40000rpm, 4degC, 1h). The protein concentration was then adjusted with cold Buffer 8 to 1-1.5mg.ml. The protein was then dialyzed against 25 volumes of refolding buffer at 4deg C (2x8h). The protein was then centrifuged (35000rpm, 1h), the supernatant was treated with GSH/GSSG (1mM/0.2mM) at 4degC overnight and concentrated before being purified further by gel filtration chromatography using a Superose 12 column.
Refolding Assay	HPLC,Crystallography,Mass spectrometry,Sequence Analysis
Refolding Chaperones	None
Refolding Additives	L-Arginine
Additives Concentration	0.65
Refolding Yield	30%, 45mg per L
Purity	95%
Notes	11 different constructs expressed and refolded, 2 different refolding buffers tried, the construct and refolding buffer reported here are the most successful. This study partly based on previous results by Chen and Gouaux (PNAS, 1997) - see record 6e9u1.

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