Refold - Collagenase 3 Also known as Matrix metalloproteinase-13

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Collagenase 3 Also known as Matrix metalloproteinase-13
Abbreviated Name	MMP-13
SCOP Family	Matrix metalloproteases, catalytic domain
Structure Notes
Organism	Mouse
UniProt Accession	P33435
SCOP Unique ID	n/a
Structure Solved
Class	Alpha+Beta
Molecularity	Unknown

Construct
Full Length	n
Domain	catalytic domain (Glu 104 to Gly 268)
Chimera	n/a
Variants	n/a
Chain Length	166
Molecular Weight	18629.7
Pi	5.16
Molecular Weight	18629.7
Disulphides	Unknown
Full Sequence	EYNVFPRTLKWSQTNLTYRIVNYTPDMSHSEVEKAFRKAFKVWSDVTPLNFTRIYDGTADIMISFGTKEHGDFYPFDGPSGLLAHAFPPGPNYGGDAHFDDDETWTSSSKGYNLFIVAAHELGHSLGLDHSKDPGALMFPIYTYTGKSHFMLPDDDVQGIQFLYG
Notes	n/a

Expression
Report	Lemaître V, Jungbluth A, Eeckhout Y. (1997) Biochem Biophys Res Commun., 230, 202-205
Project Aim	Recombinant Protein Expression
Fusion	None
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	pET-3d
Expression Protocol	Expression of CCD in E. coli. Competent E. coli BL21(DE3) were transformed with pET-CCD or with pET-3d for control. These bacteria contain a chromosomal copy of the T7 RNA polymerase which is under the control of an inducible lacUV5 promoter (14). In order to express recombinant CCD, 400 ml of an overnight culture of transformed BL21(DE3) were added to 400 ml of Luria-Bertani medium (10 g/l bactotryptone, 10 g/l NaCl, 5 g/l yeast extract) containing ampicillin (100 mg/ml). Bacteria were cultured at 37􏰆C under vigorous supplemented with leupeptin and aprotinin (2 mg/ml each). Bacteria were then lysed by two passages through a French press at 14,000 psi and inclusion bodies were recovered by centrifugation at 8,000 1 g for 10 min at 4C.
Method of Induction	IPTG
Cell Density at Induction	OD 0.6 = 600
Cell Disruption Method	French Press
Lytic Agent	None
Pre-Refolding Purification	SDS-PAGE
Solubility	insoluble

Refolding
Refolding Method	Dilution/Dialysis combination
Wash Buffer	50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl , 3 mM NaN , 1 mM ZnCl
Solubilization Buffer	50 mM Tris-HCl, pH 7.5 containing 6 M urea
Refolding Buffer	10 mM Tris-HCl, pH 7.5, 150 mM NaCl and 3 mM NaN3
Pre-Refolding Purification	SDS-PAGE
Tag Cleaved	no tag
Refolding pH	8.8
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Puriﬁcation of the recombinant CCD. Inclusion bodies from 100 ml of culture were solubilized in 3 ml of 50 mM Tris-HCl, pH 7.5 containing 6 M urea. After centrifugation at 24,000 1 g for 20 min at 4􏰆C, an equal volume of sample buffer (250 mM Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 0.005% (w/v) bromophenol blue) was added. The recombinant CCD was puriﬁed and renatured by preparative polyacrylamid gel electrophoresis (PrepCell 492, BioRad) using a 100 ml separating gel (15% acrylamid). Proteins were eluted in 25 mM Tris, 192 mM glycine, pH 8.8 at 4􏰆C. Fractions The catalytic domain of mouse collagenase-3 (CCD) containing the puriﬁed CCD were pooled and dialyzed three times against 20 volumes of 10 mM Tris-HCl, pH 7.5, 150 mM NaCl and 3 mM NaN.
Refolding Assay	Enzyme activity,Protein activity assay
Refolding Chaperones	None
Refolding Additives	Glycine
Additives Concentration	192 mM
Refolding Yield	n/a
Purity	n/a
Notes	n/a