Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Ribonuclease pancreatic
Abbreviated Name	RNase A
SCOP Family	Unknown
Structure Notes
Organism	Bovine
UniProt Accession	P61823
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	124
Molecular Weight	13690.3
Pi	8.63
Molecular Weight	13690.3
Disulphides	3
Full Sequence	KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV
Notes	n/a

Expression
Report	Goto M, Hashimoto Y, Fujita T, Ono T, Furusaki S (2000) Biotechnol Prog, 16, 1079-1085
Project Aim	Protein refolding
Fusion	None
Protein Expression and Production	Protein expressed and purified in native conformation prior to denaturation and refolding.
Expression Host	None
Expression Strain	None
Expression Temp	0.0
Expression Time	0
Expression Vector
Expression Protocol
Method of Induction	Not Stated
Cell Density at Induction	OD =
Cell Disruption Method	None
Lytic Agent	None
Pre-Refolding Purification	None
Solubility

Refolding
Refolding Method	Reversed Micelles
Wash Buffer	0.1 M Tris-HCl buffer (pH 8.5) containing denaturants 8 M urea and 0.3 M -mercaptoethanol
Solubilization Buffer	0.1 M Tris-HCl buffer, (pH 6.0-10.4, 1 mM EDTA (ethylenediaminetetraacetic acid)) into a 50-400 mM AOT-isooctane solution
Refolding Buffer	Various amount of GSH and GSSG were dissolved in a 0.1 M Tris-HCl buffer (pH 8.7, 1 mM EDTA)
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	8.7
Refolding Temperature	20.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	GSH/GSSG
Redox Agent Concentration	n/a
Refolding Protocol	Solubilization of Solid Denatured RNase A into Reversed Micelles by the Solid-Liquid Extraction Technique. A reversed micellar solution was prepared by injecting a 0.1 M Tris-HCl buffer, (pH 6.0-10.4, 1 mM EDTA (ethylenediaminetetraacetic acid)) into a 50-400 mM AOT-isooctane solution. The solid denatured RNase A was dissolved into the reversed micellar solution under ultrasonic irradiation for approximately 1 h. The concentration of RNase A solubilized in the reversed micellar solution was determined by measuring its absorbance at 278 nm with a UV-vis spectrophotometer (U-best 570 JASCO). The average diameter of reversed micelles containing proteins was analyzed by a DLS-7000 system (75 mW Ar or He/Ne laser, Otsuka Electric Co. Ltd., Japan). The obtained values are apparent radii. Renaturation of RNase A in Reversed Micelles. Various amount of reduced glutathione (GSH) and oxidized glutathione (GSSG) were dissolved in a 0.1 M Tris-HCl buffer (pH 8.7, 1 mM EDTA). The aqueous solution (1 mL) was injected into 10 mL of 400 mM AOT-isooctane solution. Renaturation was initiated by adding 1 mL of reversed micellar solution containing glutathione into the 2 mL reversed micellar solution containing solubilized RNase A. The mixture was incubated at 20 C. The specific activity of RNase A in the reversed micelles was measured against time progress, considering the time when glutathione solution was added as the initial time. The renaturation yields of denatured RNase A was evaluated by comparing the enzymatic activity of denatured RNase A to that of native RNase A that was solubilized in the reversed micelles as a control experiment.
Refolding Assay	enzyme activity
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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