Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Fas ligand interfering protein
Abbreviated Name	FIP
SCOP Family	Unknown
Structure Notes
Organism	Rat
UniProt Accession	P36940
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	278
Molecular Weight	31140.0
Pi	9.31
Molecular Weight	31140.0
Disulphides	1
Full Sequence	MQQPVNYPCPQIYWVDSSATSPWAPPGSVFSCPSSGPRGPGQRRPPPPPPPPSPLPPPSQPPPLPPLSPLKKKDNIELWLPVIFFMVLVALVGMGLGMYQLFHLQKELAELREFTNHSLRVSSFEKQIANPSTPSETKKPRSVAHLTGNPRSRSIPLEWEDTYGTALISGVKYKKGGLVINEAGLYFVYSKVYFRGQSCNSQPLSHKVYMRNFKYPGDLVLMEEKKLNYCTTGQIWAHSSYLGAVFNLTVADHLYVNISQLSLINFEESKTFFGLYKL
Notes	n/a

Expression
Report	Wisniewski P, Master A, Kaminska B. (2008) Acta Biochemicia Polonica, 55, 1
Project Aim	Cloning and expression
Fusion	N-terminal hexahistidine tag +c terminal solubilization enhancing domain (SED)
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	DE3
Expression Temp	37.0
Expression Time	3 h
Expression Vector	pET28a-m
Expression Protocol	Escherichia coli (Rosetta strain DE3, Novagen) were transformed by electroporation and 200 mL of LB medium supplemented with 30 ug/mL of kanamycin was inoculated in a 1 : 20 ratio. The bacteria were cultured at 37°C, 150 rpm until OD 600 reached 0.6. FasL Interfering Protein expression was induced by 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). After a 3 h incubation bacteria were collected by centrifugation.
Method of Induction	IPTG
Cell Density at Induction	OD 0.6 = 600
Cell Disruption Method	Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	IMAC
Solubility	insoluble

Refolding
Refolding Method	Dilution/Dialysis combination
Wash Buffer	50 mM TRIS/HCl buffer (pH 8.0) containing 10 mM β-mercaptoethanol, 1 mM EDTA and 10 0000 U/mL lysozyme
Solubilization Buffer	100 mM phosphate buffer pH 7.5 supplemented with 8 M urea
Refolding Buffer	50 mM phosphate buffer pH 7.5 containing 1 mM EDTA
Pre-Refolding Purification	IMAC
Tag Cleaved	no
Refolding pH	7.5
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	overnight
Redox Agent	Beta-mercaptoethanol
Redox Agent Concentration	10 mM,10 mM
Refolding Protocol	Isolation of inclusion bodies and protein purification. Cell pellet was suspended in 10 mL of cold 50 mM TRIS/HCl buffer (pH 8.0) containing 10 mM β-mercaptoethanol, 1 mM EDTA and 10 0000 U/mL lysozyme, and kept for 30 min on ice. Genom- ic DNA was fragmented by sonication for 5 × 20 s (Digital Sonifier 250, Branson). Crude extract was centrifuged at 14500 × g, 20 min at 4°C. Pellet was dissolved at room temperature in 100 mM phos- phate buffer pH 7.5 supplemented with 8 M urea. Denatured preparation was centrifuged at 14500 × g, 20 min, 4°C and the supernatant was subjected to IMAC chromatography using 1.25 mL of a nickel resin (His-Select, Sigma). The column was equilibrat- ed with 100 mM phosphate buffer pH 7.5 containing 8 M urea. Proteins were eluted in a linear gradient of imidazole (0–250 mM) and 33 fractions of 2 mL were collected. Fractions characterized by the highest amount of protein (Abs 280 > 0.5) were pooled. Renaturation, fluorometric assay and mass spectrometry analysis. The FIP preparation was diluted to a concentration of 70 μg/mL, and subsequently dialyzed overnight at 4°C against 50 mM phosphate buffer pH 7.5 containing 1 mM EDTA and 10 mM β-mercaptoethanol. The obtained preparation was dialyzed against phosphate saline buffer (PBS) under the same conditions. Precipitated proteins were removed by centrifugation at 170 × g (1000 rpm in MPW 370 centrifuge, rotator nr 12108), 5 min at 4 c.
Refolding Assay	Fluorescence
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	4 mg/ 1L
Purity	n/a
Notes	n/a

Back to refolding records...