Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Regulator of ribonuclease activity A (gene RV3853)
Abbreviated Name	menG
SCOP Family	RraA-like
Structure Notes
Organism	Mycobacterium tuberculosis
UniProt Accession	P0A666
SCOP Unique ID	n/a
Structure Solved
Class	Alpha/Beta
Molecularity	Trimer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	157
Molecular Weight	16235.4
Pi	5.0
Molecular Weight	16235.4
Disulphides	Unknown
Full Sequence	MAISFRPTADLVDDIGPDVRSCDLQFRQFGGRSQFAGPISTVRCFQDNALLKSVLSQPSAGGVLVIDGAGSLHTALVGDVIAELARSTGWTGLIVHGAVRDAAALRGIDIGIKALGTNPRKSTKTGAGERDVEITLGGVTFVPGDIAYSDDDGIIVV
Notes	n/a

Expression
Report	Johnston JM, Arcus VL, Morton CJ, Parker MW, Baker EN. (2003) J Bacteriol., 185, 4057-4065
Project Aim	Structural Studies
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	0.0
Expression Time	00
Expression Vector	pET42a
Expression Protocol	Cloning and expression. Open reading frame Rv3853 was amplified by PCR from genomic DNA of the H37Rv strain of M. tuberculosis. Primers were chosen that introduced a 5′ NcoI restriction site and a 3′ HindIII site. The gene was subcloned into two expression vectors: pET42a-rTEV, which gives rise to a fusion protein in which glutathione S-transferase is attached to the N terminus of the protein of interest, joined by a cleavable linker; and pProEX Hta, which gives a protein with a cleavable N-terminal poly-His tag. Expression tests in E. coli BL21(DE3)/pRI592 showed that for both constructs, the expressed protein was insoluble in all conditions tested.
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	Metal affinity chromatography
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	n/a
Solubilization Buffer	9 M urea at pH 8.0
Refolding Buffer	100 mM l-arginine, 100 mM sucrose, 50 mM morpholineethanesulfonic acid [MES], 10 mM NaCl, 0.4 mM KCl, 1 mM EDTA, 1 mM dithiothreitol
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	DTT
Redox Agent Concentration	1 mM
Refolding Protocol	Protein refolding and purification. The N-terminally His-tagged fusion protein was denatured by cell lysis in a phosphate-Tris buffer containing 9 M urea at pH 8.0 and purified by Ni2+ affinity chromatography. The protein was then refolded by dialysis at room temperature through sequential transfers, first into refolding buffer (100 mM l-arginine, 100 mM sucrose, 50 mM morpholineethanesulfonic acid [MES], 10 mM NaCl, 0.4 mM KCl, 1 mM EDTA, 1 mM dithiothreitol) and then into storage buffer (50 mM Tris-HCl [pH 8.0], 50 mM NaCl, 1 mM EDTA). The refolded protein was purified by size exclusion chromatography (Superdex 200; Pharmacia) and then further purified by anion exchange chromatography (Mono Q; Pharmacia). Light-scattering data for the final protein solution (2 mg of Rv3853 per ml) showed the protein to be a monodisperse solution of trimeric protein. The molecular mass calculated from the hydrodynamic radius was 64.5 kDa, compared with a monomer molecular mass for the His-tagged protein of 19.4 kDa.
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	L-Arginine
Additives Concentration	100 mM
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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