Refolding Record:

Protein See all refolding records for this protein...
Protein Name	CFP21
Abbreviated Name	CFP21
SCOP Family	Cutinase
Structure Notes
Organism	Mycobacterium tuberculosis
UniProt Accession	P63879
SCOP Unique ID	n/a
Structure Solved
Class	Alpha+Beta
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	220
Molecular Weight	21782.4
Pi	5.52381
Molecular Weight	21782.4
Disulphides	Unknown
Full Sequence	MTPRSLVRIVGVVVATTLALVSAPAGGRAAHADPCSDIAVVFARGTHQASGLGDVGEAFV DSLTSQVGGRSIGVYAVNYPASDDYRASASNGSDDASAHIQRTVASCPNTRIVLGGYSQG ATVIDLSTSAMPPAVADHVAAVALFGEPSSGFSSMLWGGGSLPTIGPLYSSKTINLCAPD DPICTGGGNIMAHVSYVQSGMTSQAATFAANRLDHAG
Notes	n/a

Expression
Report	Unpublished (2010) Unpublished, 99, 999
Project Aim	Undefined
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21
Expression Temp	37.0
Expression Time	4 hrs
Expression Vector	pET23b
Expression Protocol	Not stated
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	Affinity chromotography
Solubility	insoluble

Refolding
Refolding Method	Dilution/Dialysis combination
Wash Buffer	100 mM NaH2PO4, 10 mM Tris·Cl, 8 M urea pH 6.3
Solubilization Buffer	100 mM NaH2PO4, 10 mM Tris·Cl, 8 M urea pH 8.0
Refolding Buffer	100 mM NaH2PO4, 10 mM Tris·Cl, 8-0 M urea pH 8.0
Pre-Refolding Purification	Affinity chromotography
Tag Cleaved	no
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Purification of recombinant CFP21 was carried out by affinity chromatography on a Ni–NTA agarose column with certain modifications. The column was washed with water and equilibrated with buffer A (8M urea in100mM phosphate buffer, pH 8, and 10mM Tris–HCl, pH 8). The cells from 50 ml culture were suspended in 6ml buffer A and kept in shaking condition at 37°C for 2 hrs. The cell lysate containing the urea-solubilized proteins was centrifuged at 12,000g for 30 min to remove the debris and insoluble material and the supernatant was gently stirred with 2ml Ni–NTA agarose resin for 1 h at room temperature. The mixture was loaded onto a column previously equilibrated with buffer A. Briefly; the column was washed with buffer B (8M urea in 100mM phosphate buffer, pH 6.8, 10mM Tris–HCl, pH 6.8). Finally, the recombinant CFP21 was eluted with buffer C (8M urea in 100mM phosphate buffer, pH 5.9, 10mM Tris–HCl, pH 5.9) and buffer D (8M urea in 100mM phosphate buffer, pH 4.5, 10mM Tris–HCl, pH 4.5). The sample fractions were analyzed by SDS–PAGE. The fractions containing the recombinant protein were pooled and dialyzed against a decreasing concentration of urea and finally against several changes of phosphate-buffered saline (PBS) and stored in small aliquots at –20oC.
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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