Refolding Record:

Protein See all refolding records for this protein...
Protein Name	C4b-binding protein alpha chain also known as Proline-rich protein
Abbreviated Name	C4BP
SCOP Family	Unknown
Structure Notes
Organism	Human
UniProt Accession	P04003
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Dimer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	125
Molecular Weight	13836.6
Pi	6.31
Molecular Weight	13836.6
Disulphides	Unknown
Full Sequence	CGPPPTLSFAAPMDITLTETRFKTGTTLKYTCLPGYVRSHSTQTLTCNSDGEWVYNTFCIYKRCRHPGELRNGQVEIKTDLSFGSQIEFSCSEGFFLIGSTTSRCEVQDRGVGWSHPLPQCEIV
Notes	n/a

Expression
Report	Jenkins HT, Mark L, Ball G, Persson J, Lindahl G, Uhrin D, Blom AM, Barlow PN. (2006) Biologycal Chemistry, 281, 3690-3697
Project Aim	Structural Studies
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	30.0
Expression Time	5 h
Expression Vector	pET16
Expression Protocol	Human C4BP CCP1-2 (C4BP12) cDNA was amplified by PCR yielding the protein sequence: MNCGPPPTLSFAAPMDITLTETRFKTGTTLKYTCLPGY-VRSHSTQTLTCNSDGEWVYNTFCIYKRCRHPGELRNGQVEIKT-DLSFGSQIEFSCSEGFFLIGSTTSRCEVQDRGVGWSHPLPQCEILE-HHHHHH. This was cloned in the Bluescript vector using the PCR-Script cloning kit (Stratagene, La Jolla, CA) and transferred into pET16 vector (Novagen, Merck Biosciences, Nottingham, UK) using NdeI and XhoI. The DNA was transfected into BL21 (DE3) CodonPlus-RP Escherichia coli, which were cultured in Luria-Bertani broth containing kanamycin and chloramphenicol (37 °C). After cooling (30 °C), expression was induced with isopropyl--D-thiogalactopyranoside and bacteria grown for 5 h.
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Sonication
Lytic Agent	Other
Pre-Refolding Purification	Ni-NTA agrose chromatography
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	n/a
Solubilization Buffer	6 M guanidine HCl, 20 mM Tris-HCl, pH 8.0, 10 mM reduced glutathione
Refolding Buffer	55 mM Tris-HCl, pH 8.5, 10.6 mM NaCl, 2.2 mM CaCl2, 2.2 mM MgCl2, 0.055% polyethyleneglycol-4000, 0.55 M arginine, 0.1 mM oxidized glutathoine, 1 mM reduced glutathione
Pre-Refolding Purification	Ni-NTA agrose chromatography
Tag Cleaved	no
Refolding pH	8.5
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	overnight
Redox Agent	GSH/GSSG
Redox Agent Concentration	1/0.1 mM
Refolding Protocol	The sonicate was centrifuged and the pellet resuspended in 6 M guanidine HCl, 20 mM Tris-HCl, pH 8.0, 10 mM reduced glutathione. The crude material was applied to a nickel-nitrilotriacetic acid Superflow column (100 ml, Qiagen), and the protein eluted with the same guanidinium buffer plus 0.7 M imidazole. Refolding conditions (from Heiring and Muller (28)) were screened using a conformation-dependent monoclonal antibody and buffer 11 (55 mM Tris-HCl, pH 8.5, 10.6 mM NaCl, 2.2 mM CaCl2, 2.2 mM MgCl2, 0.055% polyethyleneglycol-4000, 0.55 M arginine, 0.1 mM oxidized glutathoine, 1 mM reduced glutathione) selected. Pooled fractions containing C4BP12 were diluted (20 mg/liter) in buffer 11 and incubated overnight. Iodoacetamide was added to 5 mM, and the solution incubated (30 min, 4 °C) and dialyzed against 50 mM Tris-HCl, pH 8.5. The protein solution was then applied to an 10-ml SourceQ column (Amersham Biosciences, Uppsala, Sweden) and correctly folded C4BP12 eluted with 50 mM sodium phosphate buffer, pH 8.5. 15N and 13C, 15N labeling was achieved using M9 medium supplemented with 15NH4Cl and [13C]glucose.
Refolding Assay	NMR analysis
Refolding Chaperones	None
Refolding Additives	L-Arginine
Additives Concentration	0.55 M
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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