Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Erythromycin resistance protein also known as rRNA adenine N-6-methyltransferase
Abbreviated Name	ErmSF
SCOP Family	Unknown
Structure Notes
Organism	Streptomyces fradiae
UniProt Accession	P45439
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	320
Molecular Weight	35527.5
Pi	11.6
Molecular Weight	35527.5
Disulphides	Unknown
Full Sequence	MARAPRSPHPARSRETSRAHPPYGTRADRAPGRGRDRDRSPDSPGNTSSRDGGRSPDRARRELSQNFLARRAVAERVARLVRPAPGGLLLEVGAGRGVLTEALAPYCGRLVAHEIDPRLLPALRDRFGGPHHAHVRISGGDFLAAPVPREPFALAGNIPYSRTAGIVDWALRARTLTSATFVTQLEYARKRTGDYGRWSLLTVRTWPRHEWRLLGRVSRREFRPVPRVDSGILRIERRERPLLPSAALGDYHRMVELGFSGVGGSLYASLRRAHRAGPLDAAFRAARLDRSVVVAYVTPEQWLTVFRTLRPVRSRPAGR
Notes	n/a

Expression
Report	Lee HJ, Jin HJ. (2003) Mol Cell, 16, 187-93
Project Aim	Protein refolding
Fusion	C-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	37.0
Expression Time	9.5
Expression Vector	pET23b
Expression Protocol	E. coli BL21(DE3) was transformed with the expression vector, pHJJ 105, and with empty vector. The resulting E. coli cells were grown overnight at 37°C, transferred to new LB medium (10%, v/v) and incubated at 37°C for a further 1.5 h to an A600 of 0.8–1.0. In order to induce ermSF expression as an inclusion body, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 1 mM, and incubation continued for 8 h at 37°C. The cells were pelleted and resuspended in sample buffer, and the cell extract was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS- PAGE (Laemmli, 1970)]. To obtain soluble recombinant ErmSF, the incubation temperature was lowered to 22°C and the incuba- tion time increased to 18 h, as described previously (Jin and Yang, 2002).
Method of Induction	IPTG
Cell Density at Induction	OD 0.8-1.0 = 600
Cell Disruption Method	Freeze-thaw
Lytic Agent	Lysozyme
Pre-Refolding Purification	Metal affinity chromatography
Solubility	partial

Refolding
Refolding Method	Dilution/Dialysis combination
Wash Buffer	n/a
Solubilization Buffer	6 M guanidine-HCl (buffer B)
Refolding Buffer	20 mM Tris-HCl (pH 7.0), 500 mM NaCl, 1 mM EDTA, 1 mM DTT, 150 mM imidazole, 4 M guanidine-HCl
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	yes
Refolding pH	7.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	overnight
Redox Agent	DTT
Redox Agent Concentration	1 mM
Refolding Protocol	Refolding of recombinant ErmSF After purification, the recombinant protein was diluted with buffer D to final protein concentrations of 2,000, 1,500, 1,000, 750, 500, and 250 µg/ml, and dialyzed [molecular weight cut off (MWCO): 6−8 kDa, Spectrum, Rancho Dominguez, CA] against 500 ml buffer solutions with decreasing concentrations of guanidine-HCl: first, for 3 h against buffer containing 20 mM Tris-HCl (pH 7.0), 500 mM NaCl, 1 mM EDTA, 1 mM DTT, 150 mM imidazole, 4 M guanidine-HCl; then for the same time with buffer containing 2 M guanidine-HCl, followed by overnight with buffer containing 0.5 M guanidine-HCl]. Lastly, it was dialyzed for 5 h against 20 mM Tris-HCl (pH 7.0), 500 mM NaCl. After complete removal of guanidine-HCl, the solution was centrifuged at 105,000 × g for 1 h to remove unfolded protein, and the supernatant was concentrated by dialysis against 20 mM Tris-HCl (pH 7.0), 500 mM NaCl, 50% glycerol.
Refolding Assay	Circular Dichroism (uv-CD)
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	>95%
Notes	n/a

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