Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Salivary allergen 1
Abbreviated Name	Cte f 1
SCOP Family	Unknown
Structure Notes
Organism	Ctenocephalides felis (Cat flea)
UniProt Accession	Q94424
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	176
Molecular Weight	20200.4
Pi	9.24886
Molecular Weight	20200.4
Disulphides	Unknown
Full Sequence	MNYCFLVFLV YLVFAVNGED IWKVNKKCTS GGKNQDRKLD QIIQKGQQVK IQNICKLIRD KPHTNQEKEK CMKFCKKVCK GYRGACDGNI CYCSRPSNLG PDWKVSKECK DPNNKDSRPT EIVPYRQQLA IPNICKLKNS ETNEDSKCKK HCKEKCRGGN DAGCDGNFCY CRPKNK
Notes	n/a

Expression
Report	McDermott MJ, Weber E, Hunter S, Stedman KE, Best E, Frank GR, Wang R, Escudero J, Kuner J, McCall C (2000) Molecular Immunology, 37, 361-375
Project Aim	Drug Studies
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	JK115
Expression Temp	42.0
Expression Time	1-2h
Expression Vector	pLambdaCro
Expression Protocol	4L cultures of Superbroth (3.2% Difco bactotryptone, 2% Difco bacto yeast extract, 0.5% NaCl, 5.0mM NaOH, 100 microg/ml ampicillin) were inoculated with 100ml overnight cultures. Cells were grown at 32degC in a fermenter until A600 reached 10.0-20.0, expression was then induced by increasing the temperature to 42degC then incubating a further 1-2h. Cells were harvested by centrifugation (2000g, 30min, 4degC)
Method of Induction	Temperature Shift
Cell Density at Induction	OD 600 = 10-20
Cell Disruption Method	Freeze/Thaw-High Pressure Homogenization
Lytic Agent	None
Pre-Refolding Purification	Ion-exchange chromatography
Solubility	partial

Refolding
Refolding Method	Dialysis
Wash Buffer	PBS pH 7.2, 1.0% Triton X
Solubilization Buffer	8.0M urea, 15mM Na2HPO4 pH 10.7, 0.1M 2-mercaptoethanol
Refolding Buffer	PBS pH 7.2
Pre-Refolding Purification	Ion-exchange chromatography
Tag Cleaved	no tag
Refolding pH	7.2
Refolding Temperature	4.0
Protein Concentration
Refolding Time
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Harvested cells were suspended (10:1 v/w buffer:cells) in PBS pH 7.2 and passed twice through a microfluidizer at 120psi. The suspension was centrifuged (30000g, 30min). The supernatant (S1) was set aside, the pellet was resuspended again in PBS pH 7.2 (10:1 v/w) and centrifuged again. The supernatant (S2) was retained again and the pellet again was resuspended, this time in wash buffer, and centrifuged again. 90% of the protein was present in the insoluble inclusion bodies. (The soluble fractions S1 and S2 were combined and protein was purified using a SP-sepharose column). The insoluble pellet was dissolved in solubilization buffer and then centrifuged (30000g, 30min). The supernatant was adjusted to pH7.5 and loaded onto a SP-sepharose column equilibrated wtih 8M urea, 15mM Tris pH 7.5, 100mM 2-mercaptoethanol. The protein was eluted with a linear salt gradient to buffer containing 8M urea, 1M NaCl, 50mM Tris pH 7.5, 100mM 2-mercaptoethanol in 15-20 column volumes. The purified protein was dialysed against refolding buffer and then centrifuged (10000g, 60min).
Refolding Assay	HPLC
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration
Refolding Yield
Purity
Notes

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