Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Cathepsin L1 also known as papain prosegment
Abbreviated Name	n/a
SCOP Family	Papain-like Cysteine Proteinases
Structure Notes
Organism	Human
UniProt Accession	P07711
SCOP Unique ID	n/a
Structure Solved
Class	Alpha+Beta
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	219
Molecular Weight	23838.5
Pi	4.79
Molecular Weight	23838.5
Disulphides	Unknown
Full Sequence	APRSVDWREKGYVTPVKNQGQCGSCWAFSATGALEGQMFRKTGRLISLSEQNLVDCSGPQGNEGCNGGLMDYAFQYVQDNGGLDSEESYPYEATEESCKYNPKYSVANDTGFVDIPKQEKALMKAVATVGPISVAIDAGHESFLFYKEGIYFEPDCSSEDMDHGVLVVGYGFESTNNKYWLVKNSWGEEWGMGGYVKMAKDRRNHCGIASAASYPTV
Notes	n/a

Expression
Report	Gutiérrez-González LH, Rojo-Domínguez A, Cabrera-González NE, Pérez-Montfort R, Padilla-Zúñiga AJ. (2006) Archives Biochemistry and Biophysics, 446, 151-160
Project Aim	Undefined
Fusion	None
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	3 h
Expression Vector	pET3a
Expression Protocol	The papain prosegment sequence was obtained using T7 polymerase with fluorescently labeled dideoxy terminators and an AB1373A automated sequencer (Applied Biosystems). The amplified DNA of the prosegment had the initiation (Met) and stop codons in the adequate positions and the translated sequence was identical to that reported in the UniProt Knowledgebase under the accession number P00784 for the proregion moiety. The papain prosegment gene was cloned into the pET3a vector (pET Systems, Novagen) using the restriction enzymes NdeI and BamHI. The resulting plasmids were used to transform BL21(DE3)pLysS cells (Novagen). Cultures were grown in LB broth at 37 °C with 0.1 mg ml−1 ampicillin and incubated to reach an A600 between 0.8 and 1.0. Protein expression was then induced by the addition of 0.1 mg ml−1 IPTG. Cells were harvested 3 h post-induction and centrifuged at 6000 rpm for 20 min.
Method of Induction	IPTG
Cell Density at Induction	OD 0.8-1.0 = 600
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	not specified
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	0.1 M Tris–HCl (pH 8) and 0.01 M EDTA, 2% Triton X-100
Solubilization Buffer	50 mM Tris–HCl (pH 8) containing 8 M guanidine–HCl
Refolding Buffer	50 mM phosphoric acid/NaOH buffer at pH 7
Pre-Refolding Purification	not specified
Tag Cleaved	no tag
Refolding pH	7.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	overnight
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Cell pellets containing the prosegment of papain obtained from batch cultures (1.0 L) were resuspended in 0.1 M Tris–HCl (pH 8) and 0.01 M EDTA at the rate of 10 ml/250 ml of cell culture. One hundred millimolar serinic protease inhibitor PMSF was added at 40 μl/20 ml of resuspended cells. The cells were then lysed with a cell sonicator (Branson 250). Recombinant prosegment was found in the insoluble material, which was collected by centrifugation at 45,000 rpm for 1 h and washed twice in the same buffer containing 2% Triton X-100. The material in the inclusion bodies was finally washed in buffer without Triton X-100 and solubilized in 50 mM Tris–HCl (pH 8) containing 8 M guanidine–HCl. Solubilized protein was then dialyzed overnight against 50 mM phosphoric acid/NaOH buffer at pH 7 and centrifuged at 45,000 rpm for 1 h. Sample homogeneity was confirmed by means of SDS–PAGE [17].
Refolding Assay	Circular Dichroism (uv-CD)
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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