Refold - RNA polymerase

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	RNA polymerase
Abbreviated Name	RNAP
SCOP Family	RNA-polymerase beta-prime
Structure Notes
Organism	Escherichia coli
UniProt Accession	P0A8T7
SCOP Unique ID	n/a
Structure Solved
Class	Multi-domain proteins (alpha and beta)
Molecularity	Monomer

Construct
Full Length	n
Domain	Beta-prime subunit
Chimera	n/a
Variants	n/a
Chain Length	222
Molecular Weight	25624.5
Pi	5.66
Molecular Weight	25624.5
Disulphides	0
Full Sequence	MARRASVHHHHHHMGHIELASPTAHIWFLKSLPSRIGLLLDMPLRDIERVLYFESYVVIEGGMTNLERQQILTEEQYLDALEEFGDEFDAKMGAEAIQALLKSMDLEQECEQLREELNETNSETKRKKLTKRIKLLEAFVQSGNKPEWMILTVLPVLPPDLRPLVPLDGGRFATSDLNDLYRRVINRNNRLKRLLDLAAPDIIVRNEKRMLQEAVDALLDNG
Notes	n/a

Expression
Report	Bergendahl, V., Anthony L. C., Heyduk, T., Burgess, R. R. (2002) Analytical Biochemistry, 307, 368-374
Project Aim	Recombinant Protein Expression,Protein Labeling
Fusion	N-terminal Hexahis tag + HMK recognition sequence
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	2h
Expression Vector	pTA133
Expression Protocol	Cells grown in LB medium with 100microg/ml ampicillin. Cultures were grown, induced and then harvested by centrifugation(8,000g, 15min). The cell pellet was then re-suspended in 10ml NTGED buffer(50 mM NaCl, 50 mM TrisHCl, pH 7.5, 5% glycerol, 0.1 mM EDTA, 0.1 mM DTT) + 10mM EDTA and 100microg/ml lysozyme. This solution was then incubated on ice for 30min, prior to sonication at 4degC. After sonication 1% (v/v)Triton X-100 was added and the sample vortexed. Inclusion bodies were then separated by centrifugation(25,000g, 15min).
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.5-0.7
Cell Disruption Method	Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Column refolding: Nickel-chelating chromatography
Wash Buffer	50 mM NaCl, 50 mM TrisHCl, pH 7.5, 5% glycerol, 0.1 mM EDTA, 0.1 mM DTT, 0.1% (v/v) Triton X-100
Solubilization Buffer	6 M GuHCl, 50 mM TrisHCl, pH 7.5, 0.1% Tween 20, 5mM imidazole
Refolding Buffer	500 mM NaCl, 50mM TrisHCl, pH, 7.5, 0.1% Tween 20
Pre-Refolding Purification	None
Tag Cleaved	no
Refolding pH	7.5
Refolding Temperature	20.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	DTT
Redox Agent Concentration	0.1 mM
Refolding Protocol	Inclusion bodies were washed with 10ml of washing buffer by a short sonication, prior to undergoing centrifugation(25,000g, 15min). The wash was then repeated with 10ml of washing buffer with 0.01% (v/v) Triton X-100. and suspension was aliquoted into 5 equal volumes, and underwent a final centrifugation at maximum speed. Supernatants were then discarded. Inclusion bodies were solubilized in 3 ml solubilization buffer and incubated at room temp. for 15min. Precipitate was then spun down in a microfuge(18,000g, 5min) and supernatant loaded on a Bio-Rad column with Ni-NTA matrix previously equilibrated with 0.5 ml solubilization buffer. Unbound protein was removed from the column by washing with 5ml solubilization buffer. Refolding occurred by washing with Refolding buffer.
Refolding Assay	Ligand Binding
Refolding Chaperones	None
Refolding Additives	Glycerol,Triton X-100
Additives Concentration	5%
Refolding Yield	n/a
Purity	n/a
Notes	n/a