Refolding Record:

Protein See all refolding records for this protein...
Protein Name	56kDa type-specific outer membrane antigen
Abbreviated Name	56kDa protein
SCOP Family	Unknown
Structure Notes
Organism	Orientia tsutsugamushi/Rickettsia tsutsugamushi
UniProt Accession	P22940
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Unknown

Construct
Full Length	n
Domain	aa.81-448 only
Chimera	n/a
Variants	n/a
Chain Length	368
Molecular Weight	40091.3
Pi	5.42
Molecular Weight	40091.3
Disulphides	Unknown
Full Sequence	MTIAPGFRAE LGVMYLRNIS AEVEVGKGKV DSKGEIKADS GGGTDTPIRK RFKLTPPQPT IMPISIADRD VGVDTDILAQ AAAGQPQLTV EQRAADRIAW LKNYAGIDYM VPDPQNPNAR VINPVLLNIT QGPPNVQPRP RQNLDILDHG QWRHLVVGVT ALSHANKPSV TPVKVLSDKI TKIYSDIKPF ADIAGIDVPD TGLPNSASVE QIQSKMQELN DVLEDLRDSF DGYMGNAFAN QIQLNFVMPQ QAQQQQGQGQ QQQAQATAQE AVAAAAVRLL NGNDQIAQLY KDLVKLQRHA GVKKAMEKLA AQQEEDAKNQ GEGDCKQQQG ASEKSKEGKG KETEFDLSMI VGQVKLYADL FTTESFSI
Notes	protein from. O.tsutsugamushi Gilliam strain used.

Expression
Report	Yang Q, Ching W-M, Jiang J, Lousteau L, Richards AL (2003) Annals of the New York Academy of Sciences, 990, 375-385
Project Aim	Drug Studies,Diagnostic studies
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21
Expression Temp	37.0
Expression Time	overnight
Expression Vector	pET24a
Expression Protocol	Cells were grown in LB medium containing 60microg/ml kanamycin. When OD600 reached 0.8-1.2, expression was induced with 0.4mM IPTG and cells were grown overnight. The cells were harvested and then resuspended in wash buffer without urea containing 0.1mM PMSF.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.8-1.2
Cell Disruption Method	Microfluidizer
Lytic Agent	None
Pre-Refolding Purification	Ion-exchange + size exclusion chromatography
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	20mM TrisHCl pH 8.0, 5mM EDTA, 2M urea
Solubilization Buffer	20mM TrisHCl pH 8.0, 5mM EDTA, 8M urea
Refolding Buffer	100mM NaCl, 20mM TrisHCl pH 8.0
Pre-Refolding Purification	Ion-exchange + size exclusion chromatography
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	overnig
Redox Agent	GSSG
Redox Agent Concentration	0.3mM
Refolding Protocol	Cells resuspended in wash buffer without urea containing 0.1mM PMSF were passed three times through a microfluidizer. The cell lysate was cooled immediately in an ice bath and centrifuged (10000g, 20min, 4degC). The pellets were washed in wash buffer and then centrifuged again (11000g, 30min, 4degC). The inclusion bodies were then dissolved in solubilization buffer at a concentration of2-5mg/ml. The solution was then centrifuged again (11000g, 30min, 4degC) and the supernatant retained. The solubilised protein was then purified using a DEAE column and was loaded, washed and eluted in 6-8M urea. The protein was then applied to a TSK P3000SW column in tandem with a TSK P4000SW column in refolding buffer. The protein was then refolded correctly by sequential dialysis in refolding buffer with sequentially reducing urea concentrations from 8M to 6M, 4M, 2M urea. Sequential dialysis was performed at 4degC overnight with two changes of buffer at each urea concentration. Oxidized glutathione was added to the 4M urea dialysis buffer to a final concentration of 0.3mM.
Refolding Assay	SDS-PAGE,Gel filtration chromatography,ELISA
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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