Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Outer membrane protein P2 Also known as Bac porin
Abbreviated Name	OMP P2
SCOP Family	Unknown
Structure Notes
Organism	Haemophilus influenzae
UniProt Accession	P20149
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Trimer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	341
Molecular Weight	37743.0
Pi	8.91
Molecular Weight	37743.0
Disulphides	Unknown
Full Sequence	AVVYNNEGTNVELGGRLSIIAEQSNSTVDNQKQQHGALRNQGSRFHIKATHNFGDGFYAQGYLETRFVTKASENGSDNFGDITSKYAYVTLGNKAFGEVKLGRAKTIADGITSAEDKEYGVLNNSDYIPTSGNTVGYTFKGIDGLVLGANYLLAQKREGAKGENKRPNDKAGEVRIGEINNGIQVGAKYDANDIVAKIAYGRTNYKYNESDEHKQQLNGVLATLGYRFSDLGLLVSLDSGYAKTKNYKIKHEKRYFVSPGFQYELMEDTNVYGNFKYERTSVDQGEKTREQAVLFVDHKLHKQLLTYIEGAYARTRTTETGKGVKTEKEKSVGVGLRVYF
Notes	n/a

Expression
Report	Dahan D, Srikumar R, Laprade R, Coulton JW. (1996) FEBS Letters, 392, 304-308
Project Aim	Protein refolding
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Bacillus subtilis
Expression Strain	IH6140
Expression Temp	37.0
Expression Time	00
Expression Vector	pKTH266
Expression Protocol	Cloning of the ompP2 gene from Hib ATCC 9795 into the expression vector pKTH288 of B. subtilis [16] has been described [15]. B. subtilis IH6140 cells (6 L) expressing Bac porin were grown at 37°C to stationary phase in 2XL broth containing kanamycin (30 ug/ml). Cells were harvested by centrifugation and inclusion bodies were prepared by amplifying the small-scale protocol [15]
Method of Induction	Not Stated
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Not stated
Lytic Agent	None
Pre-Refolding Purification	Size-exclusion chromatography
Solubility	insoluble

Refolding
Refolding Method	Dilution/Dialysis combination
Wash Buffer	n/a
Solubilization Buffer	20 mM Tris-HC1, pH 8.0, 8 M urea, 5 mM EDTA, 10 uM phenylmethyl-sulfonyl
Refolding Buffer	50 mM Tris-HC1, pH 8.0, 0.1% Zwittergent Z-3,14
Pre-Refolding Purification	Size-exclusion chromatography
Tag Cleaved	no tag
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	Purified Bac porin in 6 M urea was precipitated by dialysis against distilled water. Following centrifugation, the pellet was suspended in 50 mM Tris-HC1, pH 8.0, 1% SDS to a concentration of 1 mg/ml. The sample was boiled (10 min) and transferred to ice (15 min). Refolding was achieved by a three-step protocol that necessarily included Zwit-tergent Z-3,14, the same detergent in which Hib porin was originally isolated [20] and was found to be active in channel formation [21]: (1) SDS-denatured Bac porin was diluted 5-fold with a solution of 50 mM Tris-HC1, pH 8.0, 0.1% Zwittergent Z-3,14; (2) diluted Bac porin was lyophilized, resuspended in water to one-fifth its original volume and then dialysed for 12 h against 50 mM Tris-HC1, pH 8.0, 0.1% Zwittergent Z-3,14; (3) to ensure that the final Zwittergent concentration was 0.1%, Bac porin was bound to CM-Sepharose and eluted with 50 mM Tris-HCl, pH 8.0, 1 M KC1, 0.1% Zwittergent Z-3,14. The salt concentration was lowered by dialysing (12 h) against 50 mM Tris-HC1, pH 8.0, 0.1% Zwittergent Z-3,14. In attempting to refold Bac porin, several protocols were devised. Many protocols yielded \'misfolded\' Bac porin, a product which was included as a control for some experiments. Misfolded Bac porin was generated as follows: Bac porin, denatured in 50 mM Tris-HC1, pH 8.0, 2% SDS and at a concentration of 5 mg/ml, was diluted 5-fold with a solution of 50 mM Tris-HC1, pH 8.0, 0.1% Zwittergent Z-3,14. Dialysis (12 h) against 50 mM Tris-HCl, pH 8.0, 0.1% Zwittergent Z-3,14 served to reduce the amounts of SDS.
Refolding Assay	Western Blot,ELISA,Circular Dichroism (uv-CD)
Refolding Chaperones	None
Refolding Additives	Zwittergent Z-3,14
Additives Concentration	0.1%
Refolding Yield	n/a
Purity	n/a
Notes	Refolding temperature is not stated

Back to refolding records...