Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Cysteine proteinase
Abbreviated Name	CP
SCOP Family	Unknown
Structure Notes
Organism	Bombyx mori
UniProt Accession	Q26425
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	350
Molecular Weight	39657.2
Pi	6.33
Molecular Weight	39657.2
Disulphides	0
Full Sequence	MGHHHHHHHHHHSSGHIEGRHM VQFF DLVKEEWSAF KLQHRLNYKS EVEDNFRMKI YAEHKHIIAK HNQKYEMGLV SYKLGMNSWW EHGDMLHHEF VKTMNGFNKT AKHNKNLYMK GGSVRGAKFI SPANVKLPEQ VDWRKHGAVT DIKDQGKCGS CWSFSTTGAL EGQHFRQSGY LVSLSEQNLI DCSEQYGNNG CNGGLMDNAF KYIKDNGGID TEQAYPYEGV DDKCRYNPKN TGAEDVGFVD IPEGDEQKLM EAVATVGPVS VAIDASHTHF QLYSSGVYNE EECSSTDLDH GVLVVGYGTD EQGVDYWLVK NSWGRSWGEL GYIKMIRNKN NRCGIASSAS YPLV
Notes	full length protein minus signal (pre) sequence

Expression
Report	Yamamoto Y, Watabe S, Kageyama T, Takahashi SY (1999) Archives of Insect Biochemistry and Physiology, 42, 167-178
Project Aim	Structure-Function
Fusion	N-terminal hexahis + pro seq
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	3h
Expression Vector	pET16b
Expression Protocol	Cells were grown in LB broth with 50microg/ml ampicillin and 30microg/ml chloramphenicol at 37degC. Expression was induced with 1mM IPTG when A600 reached 0.6. After 3h, cells were harvested by centrifugation (4000g, 10min).
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.6
Cell Disruption Method	Chemical
Lytic Agent	Lysozyme
Pre-Refolding Purification	Metal affinity chromatography
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	50mM TrisHCl, 1mM EDTA, 100mM NaCl, 0.1mM PMSF, 0.5% Triton X-100, 10mM EDTA pH 8.0
Solubilization Buffer	1: 6M GdnHCl, 5mM imidazole, 500mM NaCl, 20mM TrisHCl pH 7.9
Refolding Buffer	50mM potassium phosphate, 5mM EDTA, 1mM GSH, 0.1mM GSSG, pH 10.7
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	yes
Refolding pH	10.7
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	overnig
Redox Agent	GSH/GSSG
Redox Agent Concentration	1mM/0.1mM
Refolding Protocol	Cell pellets were washed with cold TBS (20mM TrisHCl, 0.15M NaCl pH 7.4) and suspended in lysis buffer (50mM TrisHCl, 1mM EDTA, 100mM NaCl, 0.1mM PMSF pH 8.0). Cell lysis was started by the addition of 300microg/ml lysozyme and 0.1% deoxycholic acid and incubation was continued at 37degC until the lysate became viscous. The lysate was then further incubated at 37degC for 20min after the addition of 10microg/ml DNase 1. Inclusion bodies were isolated by centrifugation (25000g, 20min) and the pellets were then resuspended in wash buffer. The suspension was held at room temperature for 10min, then the inclusion bodies were recovered by centrifugation. Inclusion bodies were solubilized with 10ml solubilization buffer 1 and then diluted with 20ml of 9M urea. After centrifugation (20min, 25000g), the supernatant was applied to a His-Bind resin column. Following washing, the protein was eluted with 400mM imidazole, 200mM NaCl, 6M urea and 10mM TrisHCl pH 7.9. The protein was then solubilized with solubilization buffer 2 at 100mg/ml and incubated at 56degC for 2h. The protein was centrifuged (20min, 25000g) and the supernatant was refolded by being slowly added dropwise into 200 volumes of refolding buffer and stirred overnight at 4degC. The pH was adjusted to pH 8.0 with HCl, then the protein was centrifuged again (20min, 25000g) and the supernatant containing the refolded protein retained.
Refolding Assay	Enzyme activity
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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