Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Chitinase ECH30
Abbreviated Name	ech30
SCOP Family	Unknown
Structure Notes
Organism	Trichoderma atroviride
UniProt Accession	Q56VR5
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	309
Molecular Weight	32493.9
Pi	3.93
Molecular Weight	32493.9
Disulphides	Unknown
Full Sequence	MYFRKAAAVTSLLAALSSAQPSGPELAVYWGAEDDSTTLNDVCSDSSYGIVNLAFLDTFFAAGGFPQLSLSGLDGPSQAQQSAGATGLKDGSSLVDAIQQCQSAGKLVLLSLGGAGADVTLQSDSDGEKIADTLWNLFGGGTDNQELRPFGDIKLDGFDLDNESGNPTGYLAMVQRFKSNFQNDTSKSYFLSAAPQCPFPDASQSQDVCSELDFVWVQFYNNGDCNIAQCDFLNSVQTWSSGIGNAKLYIGALASGADGDQGFADANTLLGAIQDVKNMNLPNYAGAMLWEAQLAVQNGNFQQKIAPGL
Notes	n/a

Expression
Report	Hoell IA, Klemsdal SS, Vaaje-Kolstad G, Horn SJ, Eijsink VG. (2005) Biochemica et Biophysica Acta, 1748, 180-190
Project Aim	Expression and charactrization
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(DE3)
Expression Temp	37.0
Expression Time	4 h
Expression Vector	pETM10
Expression Protocol	After a series of initial experiments (see Results section) plasmid pETM10-ech30, containing the ech30 gene without signal peptide and with N-terminal (His)6-tag, was selected for further studies. E. coli BL21Star (DE) transformants containing the pETM10-ech30 construct were grown at 37 °C in LB-medium with 50 μg ml−1 kanamycin at 225 rpm, to a cell density of 0.6 at 600 nm. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM, and the cells were further incubated for 4 h at 37 °C followed by harvesting by centrifugation (8000 rpm, 8 min at 4 °C).
Method of Induction	IPTG
Cell Density at Induction	OD 0.6 = 0.6
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Ni-NTA column
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	N/a
Solubilization Buffer	8 M urea, 0.1 M NaH2PO4 and 0.01 M Tris–HCl, pH 8.0
Refolding Buffer	20 mM Tris–HCl, pH 8.0
Pre-Refolding Purification	Ni-NTA column
Tag Cleaved	yes
Refolding pH	8.0
Refolding Temperature	0.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	The cell pellet was resuspended in citrate–phosphate buffer pH 6.0, and the cells were lysed by sonication at 20% amplitude with 30×5 s pulses (with 5 s delay between pulses) on ice, with a Vibra cell Ultrasonic Processor, converter model CV33, equipped with a 3 mm probe (Sonics, Newtown, CT, USA). The inclusion bodies were harvested by centrifugation (15 000 rpm, 20 min at 4 °C) and solubilized in 8 M urea, 0.1 M NaH2PO4 and 0.01 M Tris–HCl, pH 8.0. Non-solubilizable material was removed by centrifugation (15 000 rpm, 10 min at room temperature). The solubilized protein was purified under denaturing conditions (with 8 M urea present) on a 10×60 mm Ni-NTA column (Qiagen, Venlo, The Netherlands) using a flow rate of 2 ml/min. The column was equilibrated in citrate–phosphate buffer pH 6.0, containing 8 M urea and 20 mM imidazole. After loading the solubilized protein, the column was washed with the starting buffer. The His-tagged protein was then eluted with a citrate–phosphate buffer pH 6.0, containing 8 M urea and 100 mM imidazole, and the purified and solubilized protein was refolded by dialysis against 20 mM Tris–HCl, pH 8.0. This procedure enabled production of milligrams amounts of enzyme (approximately 15–20 mg refolded Ech30 pr. liter culture).
Refolding Assay	enzyme activity
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	15–20 mg/L
Purity	n/a
Notes	n/a

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