Refold - Cell division control protein 42 homolog

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Cell division control protein 42 homolog
Abbreviated Name	Cdc42
SCOP Family	G proteins
Structure Notes
Organism	Human
UniProt Accession	P60953
SCOP Unique ID	n/a
Structure Solved
Class	Alpha/Beta
Molecularity	Monomer

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	isoform 2
Chain Length	213
Molecular Weight	24056.8
Pi	9.06
Molecular Weight	24056.8
Disulphides	0
Full Sequence	MHHHHHHYGRKKRRQRRRMGGGMQTIKCVVVGDGAVGKTCLLISYTTNKFPSEYVPTVFDNYAVTVMIGGEPYTLGLFDTAGQEDYDRLRPLSYPQTDVFLVCFSVVSPSSFENVKEKWVPEITHHCPKTPFLLVGTQIDLRDDPSTIEKLAKNKQKPITPETAEKLARDLKAVKYVECSALTQKGLKNVFDEAILAALEPPEPKKSRRCVLL
Notes	n/a

Expression
Report	Becker-Hapak, M., McAllister, S. S., Dowdy, S. F. (2001) Methods, 24, 247-256
Project Aim	Protein Transduction
Fusion	N-terminal hexahis + TAT sequence
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)pLysS
Expression Temp	37.0
Expression Time	6h
Expression Vector	pET-TAT-HA
Expression Protocol	1L LB broth containing 50microg/ml ampicillin was inoculated with 200ml overnight culture, and induced and allowed to incubate at 225rpm. Cells were harvested by centrifugation(5000g, 10min) and resuspended in 20 ml of buffer z(8M urea, 100mM NaCL, 20mM Hepes, pH 8.0). Cells were then lyzed (4x12s on, 15 off at 4degC) and clarified by centrifugation(16,000g, 10sec, 4degC).
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Metal affinity chromatography
Solubility	not stated

Refolding
Refolding Method	column refolding: ion-exchange chromatography
Wash Buffer	n/a
Solubilization Buffer	4M Urea, 50mM NaCl, 20 mM Hepes, pH 8.0
Refolding Buffer	50mM NaCl, 20mM Hepes, pH 8.0
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no
Refolding pH	8.0
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Dilute Ni-NTA fraction from above 1:1 with 20mM Hepes, pH 8.0 for MonoQ and 6.5 for MonoS. Inject the sample into a 5/5 or 10/10 MonoQ/S column equilibrated with buffer A(50mM NaCl, 20mM Hepes, pH 8.0) plus Urea(4 or 8M depending on temp). Wash with 20-50ml Buffer A and elute with 1-2M NaCl in buffer A. The Sample is then desalted and allowed to refold on a PD-10 column by reducing 4-0M Urea.
Refolding Assay	Fluorescence,Immunoassay,Actin Binding Activity
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a