Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Prochymosin
Abbreviated Name	Prochymosin
SCOP Family	Pepsin-like proteases
Structure Notes
Organism	Bovine
UniProt Accession	P00794
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Monomer

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	Prochymosin A variant, G244D
Chain Length	366
Molecular Weight	40666.8
Pi	4.76438
Molecular Weight	40666.8
Disulphides	3
Full Sequence	MAEIT RIPLYKGKSL RKALKEHGLL EDFLQKQQYG ISSKYSGFGE VASVPLTNYL DSQYFGKIYL GTPPQEFTVL FDTGSSDFWV PSIYCKSNAC KNHQRFDPRK SSTFQNLGKP LSIHYGTGSM QGILGYDTVT VSNIVDIQQT VGLSTQEPGD VFTYAEFDGI LGMAYPSLAS EYSIPVFDNM MNRHLVAQDL FSVYMDRNGQ ESMLTLGAID PSYYTGSLHW VPVTVQQYWQ FTVDSVTISD VVVACEGGCQ AILDTGTSKL VGPSSDILNI QQAIGATQNQ YGEFDIDCDN LSYMPTVVFE INGKMYPLTP SAYTSQDQGF CTSGFQSENH SQKWILGDVF IREYYSVFDR ANNLVGLAKA I
Notes	n/a

Expression
Report	Menzella HG, Gramajo HC, Ceccarelli EA (2002) Protein Expression and Purification, 25, 248-255
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
Expression Temp	37.0
Expression Time	6h
Expression Vector	pET22b
Expression Protocol	Cells were grown in Luria–Bertani medium containing ampicillin (100 microg/ml) at 37degC to an optical density of 0.7 at 600 nm. Protein expression was induced by adding 1 mM IPTG (final concentration) and incubation continued for an additional 6 h period. Cells were then harvested by centrifugation at 10,000g for 15 min at 4degC.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.7
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	soluble

Refolding
Refolding Method	Dilution
Wash Buffer	50 ml of 10 mM EDTA (pH 8.0), 0.5% (v/v) Triton X-100
Solubilization Buffer	8 M urea in 50 mM KH2PO4 (pH 10.5)
Refolding Buffer	50 mM KH2PO4, 10 microM CuSO4, 0.5M L-arginine
Pre-Refolding Purification	None
Tag Cleaved	yes
Refolding pH	10.5
Refolding Temperature	4.0
Protein Concentration	0.8mg.ml
Refolding Time	16h
Redox Agent	DTT
Redox Agent Concentration	1-10mM
Refolding Protocol	To assess the refolding of prochymosin using air oxidation, the unfolded protein was diluted in 50 mM KH2PO4 (pH 10.5) in the presence of 10 microM CuSO4 and 0.5M L-arginine (final concentrations). Oxidation was carried out by sparging air at a flow rate of 0.1 L/min in identical 1 L Erlenmeyer flasks and sparging systems, thus ensuring reproducibility of the oxidation conditions. Refolding of recombinant sulfonated prochymosin was performed as follows: The inclusion bodies were resuspended in 10 mM Tris–HCl (pH 8.0), 10 M urea, 0.3 M Na2SO3, and incubated at 37degC for 30 min. Sulfonation was completed by the addition of 20 mM 2-nitro-thiosulfobenzoate final concentration, followed by incubation of the solution for 15 min at room temperature in the dark. The reaction was quenched by extensive dialysis of the sulfonated recombinant prochymosin against 0.7% (v/v) acetic acid at 4degC to minimize unwanted disulfide shuffling. Sulfonated recombinant prochymosin was found to be insoluble in the absence of the denaturant. The protein precipitate was recovered by centrifugation at 10,000g for 20 min at 4degC and solubilized in 50 mM KH2PO4 (pH 10.7), urea 8 M. Refolding was started by diluting 40 ml of the unfolded protein solution (20 mg/ml) in 1 L of degassed renaturation buffer containing 50 mM KH2PO4 (pH 10.7), 1 mM EDTA, DTT (1–10 mM), and incubated at selected temperatures.
Refolding Assay	Enzyme activity
Refolding Chaperones	None
Refolding Additives	None,L-Arginine
Additives Concentration	0.5M
Refolding Yield	67%
Purity	95% prochymosin, as judged by SDS?PAGE
Notes

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