Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Merezoite surface protein 3
Abbreviated Name	MSP3
SCOP Family	Unknown
Structure Notes
Organism	Plasmodium falciparum
UniProt Accession	Q95PI5
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Tetramer

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	131
Molecular Weight	15426.2
Pi	4.15545
Molecular Weight	15426.2
Disulphides	0
Full Sequence	ISKENDDVLD EKEEEAEETE EEELEEKNEE ETESEISEDE EEEEEEEEEK EEENDKKKEQ EKEQSNENND QKKDMEAQNL ISKNQNNNEK NVKEAAESIM KTLAGLIKGN NQIDSTLKDL VEELSKYFKN H
Notes	n/a

Expression
Report	Burgess BR, Schuck P, Garboczi DN (2005) J Biol Chem, 280, 37236-37245
Project Aim	Structure-Function
Fusion	None
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	3.5h
Expression Vector	pLM1
Expression Protocol	Cells were grown until A600 reached 0.8, then 1mM IPTG was added. Cells were grown a further 3.5h, then harvested by centrifugation (8000rpm, 10min, 4degC).
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.8
Cell Disruption Method	Freeze-thaw
Lytic Agent	Lysozyme
Pre-Refolding Purification	Metal affinity chromatography
Solubility	soluble

Refolding
Refolding Method	Dialysis
Wash Buffer	n/a
Solubilization Buffer	8M urea, PBS, 2mM DTT
Refolding Buffer	PBS, 4mM DTT
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no
Refolding pH	7.4
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	24h
Redox Agent	DTT
Redox Agent Concentration	4mM,4mM
Refolding Protocol	The cells were washed with 20mM TrisHCl pH 8.0, 2mM NaEDTA, 200mM NAcl and pelleted. The cell pellets were then resuspended in 50ml of 50mM TrisHCl pH 8.0, 5mM NaEDTA, 2mM DTT, 1mM 4-(2-aminoethyl)benzenesulfonyl fluoride HCl, 0.1mg/ml lysozyme and 1 Complete Protease inhibitor tablet per L of culture with 2mM DTT present. The cells were subjected to two cycles of freeze/thaw in the presence of 10mM MgCl2 and 20microg/ml DNase. The solution was then centrifuged (12000rpm, 30min) and the supernatant retained. The supernatant was heat-treated at 80degC for 25min, followed by removal of the insoluble material by centrifugation. The supernatant was then loaded onto a Q-sepharose column and eluted in 50mM Mes-NaOH pH 5.5, 2mM DTT with a 0-600mM NaCl gradient. Selected fractions were pooled and concentrated, then passed down a Superdex200 column in PBS with 2mM. Again, selected fractions were pooled and concentrated. Crystalline urea was added to a final concentration of 8M. The protein was then dialysed at 4degC against three changes (100x volume) of refolding buffer over 24h. The protein was then concentrated and finally purified by three sequential passages down a size exclusion column.
Refolding Assay	Far-UV Circular Dichroism,Gel filtration chromatography,Sedimentation velocity analysis
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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