Refolding Record:

Protein See all refolding records for this protein...
Protein Name	PPE FAMILY PROTEIN
Abbreviated Name	Rv2430c
SCOP Family	PE
Structure Notes
Organism	Mycobacterium tuberculosis
UniProt Accession	Q79FE1
SCOP Unique ID	n/a
Structure Solved
Class	Alpha
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	194
Molecular Weight	21942.8
Pi	4.77
Molecular Weight	21942.8
Disulphides	Unknown
Full Sequence	MHFEAYPPEVNSANIYAGPGPDSMLAAARAWRSLDVEMTAVQRSFNRTLLSLMDAWAGPVVMQLMEAAKPFVRWLTDLCVQLSEVERQIHEIVRAYEWAHHDMVPLAQIYNNRAERQILIDNNALGQFTAQIADLDQEYDDFWDEDGEVMRDYRLRVSDALSKLTPWKAPPPIAHSTVLVAPVSPSTASSRTDT
Notes	n/a

Expression
Report	Choudhary RK, Pullakhandam R, Ehtesham NZ, Hasnain SE. (2004) Protein Expression and Purification, 36, 249-253
Project Aim	Recombinant Protein Expression
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	M15pREP4
Expression Temp	37.0
Expression Time	3 h
Expression Vector	pREP4
Expression Protocol	Expression and purification of the recombinant protein coded by Rv2430c The Rv2430c was cloned and expressed as previously described [16]. Recombinant plasmid PQE30Rv2430c carrying Rv2430c as a N-terminal histidine tagged fusion was transformed into the host M15pREP4 strain of E. coli and induced for expression by 1 mM IPTG. Cells were harvested 3 h post-induction. The harvested cells were suspended in buffer A (25 mM Tris–Cl, pH 8.0, containing 8 M urea and 0.9% NaCl) and incubated on an end-to-end shaker for 30 min at room temperature for lysis.
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Not stated
Lytic Agent	None
Pre-Refolding Purification	Ni-NTA agrose chromatography
Solubility	insoluble

Refolding
Refolding Method	Column refolding: Nickel-chelating chromatography
Wash Buffer	n/a
Solubilization Buffer	25 mM Tris–Cl, pH 8.0, containing 8 M urea and 0.9% NaCl
Refolding Buffer	25 mM Tris, pH 8.0, 5 mM imidazole, 1 mM glutathione, and 0.1 M -arginine hydrochloride
Pre-Refolding Purification	Ni-NTA agrose chromatography
Tag Cleaved	no
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	GSH
Redox Agent Concentration	n/a,1 mM,n/a
Refolding Protocol	The lysate was centrifuged at 13,000 rpm for 30 min and the supernatant was then incubated with pre-equilibrated Ni–NTA slurry (Qiagen, USA) for 15–20 min with gentle agitation to maximize the binding of the recombinant protein. The protein bound to slurry was then packed into a column. The bound protein was then subjected to on-column refolding by using a 250 ml gradient of buffer A and buffer B (25 mM Tris, pH 8.0, 5 mM imidazole, 1 mM glutathione, and 0.1 M -arginine hydrochloride) at a flow rate of 1 ml/min using Acta-Prime chromatographic unit (Pharmacia Biotech). At the end of the gradient, the column was further washed with 50 ml buffer B and then eluted with 25 mM Tris, pH 8.0, containing 500 mM imidazole. The homogeneity of the eluted protein was confirmed by 12% SDS–PAGE and the purified protein was dialyzed extensively at 4 °C against 25 mM Tris–HCl, pH 8.0, containing 0.9% NaCl. Protein was quantified by Pierce Micro BCA Protein Assay Reagent kit (Pierce, USA) and was subsequently used for spectroscopic analyses.
Refolding Assay	Fluorescence,Activity assay
Refolding Chaperones	None
Refolding Additives	None,L-Arginine
Additives Concentration	0.1 M
Refolding Yield	n/a
Purity	n/a
Notes

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