Refolding Record:

Protein See all refolding records for this protein...
Protein Name	DELLA protein RGL3
Abbreviated Name	RG-3
SCOP Family	Unknown
Structure Notes
Organism	Arabidopsis thaliana
UniProt Accession	Q9LF53
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	523
Molecular Weight	57326.7
Pi	4.75
Molecular Weight	57326.7
Disulphides	Unknown
Full Sequence	MKRSHQETSVEEEAPSMVEKLENGCGGGGDDNMDEFLAVLGYKVRSSDMADVAQKLEQLEMVLSNDIASSSNAFNDTVHYNPSDLSGWAQSMLSDLNYYPDLDPNRICDLRPITDDDECCSSNSNSNKRIRLGPWCDSVTSESTRSVVLIEETGVRLVQALVACAEAVQLENLSLADALVKRVGLLAASQAGAMGKVATYFAEALARRIYRIHPSAAAIDPSFEEILQMNFYDSCPYLKFAHFTANQAILEAVTTSRVVHVIDLGLNQGMQWPALMQALALRPGGPPSFRLTGVGNPSNREGIQELGWKLAQLAQAIGVEFKFNGLTTERLSDLEPDMFETRTESETLVVNSVFELHPVLSQPGSIEKLLATVKAVKPGLVTVVEQEANHNGDVFLDRFNEALHYYSSLFDSLEDGVVIPSQDRVMSEVYLGRQILNLVATEGSDRIERHETLAQWRKRMGSAGFDPVNLGSDAFKQASLLLALSGGGDGYRVEENDGSLMLAWQTKPLIAASAWKLAAELRR
Notes	n/a

Expression
Report	Taha H. Al-Samarrai, Christopher A. Kirka, William T. Jonesa, Dawn Harveya and Xiaolin Suna (2007) Protein Expression and Purification, 53, 289-292
Project Aim	Structure-Function
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	37.0
Expression Time	3h
Expression Vector	pET-21
Expression Protocol	E. coli strain BL21 (DE3) expressing His-tag (At) RGL-3, was grown in LB medium containing ampicillin (100 μg/ml) to an A600 of 0.3–0.4 at 37 °C. Expression was induced by addition of IPTG (0.3 mM) followed by 3 h incubation at 37 °C. Cells were collected by centrifugation and resuspended in a lysis buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 0.1 mM PMSF, 1 mM EDTA, and 0.5 mM dithiothreitol) and frozen at −20 °C.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.3
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	20 mM Tris–HCl, pH 8.4, 5 mM EDTA, 500 mM NaCl, 2 M urea, 2% Triton-X
Solubilization Buffer	8 M urea, 100 mM Tris–HCl, 5 mM EDTA, 10 mM dithiothreitol, 1.5 mM reduced glutathione, 0.2 mM oxidized glutathione, pH 8.4
Refolding Buffer	20 mM Tris–HCl, 50 mM NaCl, 2 mM EDTA–(Na)2, pH 8.4
Pre-Refolding Purification	None
Tag Cleaved	no
Refolding pH	8.4
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	GSH/GSSG
Redox Agent Concentration	1.5
Refolding Protocol	Refolding of (At) RGL-3-His-tag was initiated at 4 °C by 20-fold dilution of the urea-denatured proteins of inclusion bodies with buffer B (20 mM Tris–HCl, 50 mM NaCl, 2 mM EDTA–(Na)2, pH 8.4) and placed in ice-bath. The mixture was then sonicated in an ultrasonic bath for 10 min (Samophone, Ultrasonic Industries Pvt., Ltd.). The mixture was then centrifuged (19,000 rpm for 1 h at 4 °C). The supernatants were aspirated and concentrated 10 times by ultra filtration (20 kDa type 20 Diafilter Pall Filtron Technology Corporation) at 4 °C. The (At) RGL-3 was further purified by employing Ni–NTA chromatography. The concentrate was loaded onto a 5-ml Ni–NTA column equilibrated with buffer B plus 0.4 M urea and 5 mM imidazole. The column was washed with the same buffer containing 0.4 M urea and 30 mM imidazole. The His-tagged (At) RGL-3 was eluted from the column with buffer B containing 0.4 M urea and 300 mM imidazole. Fractions containing (At) RGL-3 were pooled and concentrated by ultrafiltration as above.
Refolding Assay	Native PAGE
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	87%
Purity	n/a
Notes	Further purification resulted in oligomerisation of the protein.

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