Refold - Arg specific Gingipain 1

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Arg specific Gingipain 1
Abbreviated Name	RGP-1
SCOP Family	Gingipain R (RgpB), N-terminal domain
Structure Notes
Organism	Porphyromonas gingivalis
UniProt Accession	P28784
SCOP Unique ID	n/a
Structure Solved
Class	Alpha/Beta
Molecularity	Monomer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	524
Molecular Weight	57182.5
Pi	5.94209
Molecular Weight	57182.5
Disulphides	0
Full Sequence	MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRG TPVEEKQNGRMI VIVAKKYEGD IKDFVDWKNQ RGLRTEVKVA EDIASPVTAN AIQQFVKQEY EKEGNDLTYV LLVGDHKDIP AKITPGIKSD QVYGQIVGND HYNEVFIGRF SCESKEDLKT QIDRTIHYER NITTEDKWLG QALCIASAEG GPSADNGESD IQHENVIANL LTQYGYTKII KCYDPGVTPK NIIDAFNGGI SLVNYTGHGS ETAWGTSHFG TTHVKQLTNS NQLPFIFDVA CVNGDFLFSM PCFAEALMRA QKDGKPTGTV AIIASTINQS WASPMRGQDE MNEILCEKHP NNIKRTFGGV TMNGMFAMVE KYKKDGEKML DTWTVFGDPS LLVRTLVPTK MQVTAPAQIN LTDASVNVSC DYNGAIATIS ANGKMFGSAV VENGTATINL TGLTNESTLT LTVVGYNKET VIKTINTNGE PNPYQPVSNL TATTQGQKVT LKWDAPSTKT NATTNTARSV DGIRELVLLS VSDAPELLR
Notes	n/a

Expression
Report	Margetts MB, Barr IG, Webb EA (2000) Protein Expression and Purification, 18, 262-268
Project Aim	Drug Studies
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	HMS174(DE3)
Expression Temp	37.0
Expression Time	3h
Expression Vector	pET28a
Expression Protocol	Cells in Terrific Broth containing potassium phosphates plus 50 mg/ml kanamycin were grown until OD600 reached 0.6. Expression was induced with 0.1mM IPTG and cells were grown for 3h before being harvested by centrifugation at 10,000g and the bacterial pellets resuspended to an OD600 of 8.0 in PBS and stored at -20degC.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.6
Cell Disruption Method	Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	Metal affinity chromatography
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	5mM imidazole, 500mM NaCl, 20mM TrisHCl pH 7.9
Solubilization Buffer	20mM Tris-HCl, 5mM imidazole, 500mM NaCl, 6M urea, pH 7.9
Refolding Buffer	100mM Tris-HCl, 150mM NaCl, 5mM CaCl2, 5mM DTT, 5mM cysteine, pH7.8
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no
Refolding pH	7.8
Refolding Temperature	25.0
Protein Concentration
Refolding Time
Redox Agent	DTT/cysteine
Redox Agent Concentration	5mM/5mM
Refolding Protocol	Bacterial pellets were thawed on ice and resuspended in wash buffer and then sonicated. Lysates were centrifuged at 20,000g to collect the inclusion bodies. Pellets were resuspended and the process of sonication and centrifugation was repeated twice more to release trapped proteins. Pellets were finally resuspended in solubilization buffer and incubated on ice for 2–3h with continuous stirring to solubilize proteins. Any remaining insoluble material was removed by centrifuging at 39,000g for 20 min. The supernatant was filtered through a 0.45micron membrane before column purification. The protein was then purified under denaturing conditions using a Ni-NTA column, then refolded by dialysis against refolding buffer.
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration
Refolding Yield
Purity
Notes	The final product contains contaminants originating from autolytic cleavage. Other refolding methods and conditions trialled - see paper for more details.