Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Type XI collagen alpha-1 chain
Abbreviated Name	Npp alpha1 XL
SCOP Family	Unknown
Structure Notes
Organism	Rattus norvegicus (Rat)
UniProt Accession	Q8VBY8
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	535
Molecular Weight	57396.3
Pi	5.45
Molecular Weight	57396.3
Disulphides	Unknown
Full Sequence	APVDILKALDFHNSPVGISKTTGFCTSRKNSKDPDIAYRVTEEAQISAPTKQLFPGGIFPQDFSILFTIKPKKGTQAFLLSLYNEHGIQQLGVEVGRSPVFLFEDHTGKPTPENYPLFSTVNIADGKWHRVAISVEKKTVTMIVDCKKKITKPLDRSERSIVDTNGIMVFGTRILETDVFQGDIQQFLITGDPKAAYDYCDHYSPDCDLTSKAAQAQEPHIDEKKKSNYTKKKRTLATNSKKKSKMSTTPKSEKFASKKKKRNQATAKAKLGVQANIVDDFQDYNYGTMETYQTESPRRV SGSNEPNPVEEGFTEEYLTGEDYDVQRNISEDILYGNKGIDGRDSDLLVDGDLGEYDFYEYKEYEERTTTAPNEEFGPGVPAETDFTETSINGHGAYGEKGQKGEPAVVEPGMLVEGPPGPAGPAGLMGPPGLQGPSGLPGDPGDRGPPGRPGLPGADGLPGPPGTMLMLPFRYGGDGSKGPTISAQEAQAQAILQQARIALRGPPGPMGLTGRPGPVGGPGSAGAKGESGDP
Notes	n/a

Expression
Report	Fallahi A, Kroll B, Warner LR, Oxford RJ, Irwin KM, Mercer LM, Shadle SE, Oxford JT. (2005) Protein Science, 14, 1526-37
Project Aim	Structural Studies
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)pLysS
Expression Temp	0.0
Expression Time	00
Expression Vector	pET11a
Expression Protocol	Construction of the Npp α1(XI) collagen expression vector The Npp α1(XI) collagen sequence was amplified and cloned into pET11a expression vector as previously described (Gregory et al. 2000). E. coli BL21(DE3)pLysS-competent cells were transformed and grown in LB medium. Cells were grown to early log phase and recombinant protein expression was induced by the addition of IPTG. Cells were harvested by centrifugation and lysed by resuspension in a hypotonic detergent solution (B-PER, Pierce).
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	None
Lytic Agent	Detergents
Pre-Refolding Purification	not specified
Solubility	partial

Refolding
Refolding Method	Column refolding: Nickel-chelating chromatography
Wash Buffer	phosphate buffered saline
Solubilization Buffer	50 mM Tris-HCl (pH 7.5) containing 6 M guanidine hydrochloride and 150 mM NaCl
Refolding Buffer	imidazole concentration gradient from 5 mM to 250 mM
Pre-Refolding Purification	not specified
Tag Cleaved	no tag
Refolding pH	0.0
Refolding Temperature	0.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Cells were grown to early log phase and recombinant protein expression was induced by the addition of IPTG. Cells were harvested by centrifugation and lysed by resuspension in a hypotonic detergent solution (B-PER, Pierce). Recombinant protein was collected by differential centrifugation to separate inclusion bodies from soluble proteins, followed by subsequent washes in phosphate buffered saline at 4°C until purity of the recombinant protein approached 70% as assessed by SDS-polyacrylamide gel electrophoresis. Inclusion body proteins were solubilized in 50 mM Tris-HCl (pH 7.5) containing 6 M guanidine hydrochloride and 150 mM NaCl, and clarified by centrifugation at 18,000g, 4°C for 30 min. Soluble protein was applied to a nickel-NTA affinity chromatography column at a flow rate of 1 mL/min. Protein was refolded while on the column. Absorbance at 280 nm and conductivity were monitored continuously. Bound protein was eluted using an imidazole concentration gradient from 5 mM to 250 mM. Protein in each fraction was assessed by SDS-polyacrylamide gel electrophoresis. Proper structure of folded protein was confirmed by circular dichroism and mass spectrometry as previously described (Gregory et al. 2000).
Refolding Assay	Mass spectrometry,Circular Dichroism (uv-CD)
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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