Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Proteinase inhibitor PSKP-1
Abbreviated Name	PSKP-1
SCOP Family	Unknown
Structure Notes
Organism	Phyllomedusa sauvagei (Sauvage\'s leaf frog)
UniProt Accession	P83578
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Monomer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	58
Molecular Weight	6702.0
Pi	9.34
Molecular Weight	6702.0
Disulphides	3
Full Sequence	VIEPKCYKYEGKKCPPDINPVCGTDKRTYYNECALCVFIRQSTKKADKAIKIKKWGKC
Notes	n/a

Expression
Report	Gebhard LG, Carrizo FU, Stern AL, Burgardt NI, Faivovich J, Lavilla E, Ermácora MR. (2004) Eur J Biochem., 271, 2117-2126
Project Aim	Protein isolation
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)
Expression Temp	37.0
Expression Time	3 h
Expression Vector	pET-PSKP-1
Expression Protocol	PSKP-1 encoding DNA was custom synthesized and ligated into a cloning vector by Interactiva Inc. (Ulm, Germany). Codon usage was optimized for expression in E. coli[22]. The fragment encoding recombinant PSKP-1 was subcloned into pET-15b (Novagen) at NcoI and BamHI sites. The resulting expression vector, pET-PSKP-1, was sequenced to confirm the reading frame and the identity of the insert. For expression, E. coli BL21 (DE3) cells were transformed with pET-PSKP-1 and grown to saturation [overnight at 37 °C in LB (Luria–Bertani) broth containing 100 mg·mL−1 ampicillin). The saturated culture (2 mL) was used to inoculate 1 L of fresh broth, and growth was continued to reach an attenuance (D), at 600 nm, of ≈ 1. Then, either 1 mm isopropyl thio-β-d-galactoside or 1% (w/v) lactose was added, and incubation continued for 3 h. Cells were harvested by centrifugation at 5000 g (10 min at 4 °C), and the resulting pellet was stored at −20 °C. As PSKP-1 was present in inclusion bodies, harvested cells (3–4 g) were suspended in 8 mL of lysis buffer (50 mm Tris/HCl, 100 mm NaCl, 1 mm EDTA, pH 8.0) and disrupted by sonication in an ice bath (in 4 mL fractions, five pulses of 30 s and 4 watts).
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	not specified
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	n/a
Solubilization Buffer	20 mm sodium phosphate, 8 m urea, 2 mm glycine, 50 mm dithiothreitol, pH 7.5
Refolding Buffer	20 mm sodium phosphate, 5.0 mm 2-mercaptoethanol, 0.5 mm cystamine, pH 7.5, and then exhaustively against distilled water
Pre-Refolding Purification	not specified
Tag Cleaved	no tag
Refolding pH	7.5
Refolding Temperature	5.0
Protein Concentration	n/a
Refolding Time	overnight
Redox Agent	Beta-mercaptoethanol
Redox Agent Concentration	5 mM
Refolding Protocol	Cleaned inclusion bodies were solubilized at 37 °C in buffer B (20 mm sodium phosphate, 8 m urea, 2 mm glycine, 50 mm dithiothreitol, pH 7.5) and loaded onto an SP Sepharose Fast-Flow (Pharmacia Biotech) cationic exchange column (1.5 × 5.0 cm) equilibrated with buffer B. Elution was performed with a 200 mL linear gradient of 0–0.5 m NaCl in buffer B. Fractions were monitored by absorbance at 280 nm and SDS/PAGE. Fractions containing pure PSKP-1 were dialyzed, first overnight at 5 °C against 100 volumes of 20 mm sodium phosphate, 5.0 mm 2-mercaptoethanol, 0.5 mm cystamine, pH 7.5, and then exhaustively against distilled water. Finally, particulate matter was removed by centrifugation (24 000 g, 20 min, 4 °C) and the solution fast-frozen to −80 °C and lyophilized. The resulting product was stored at −20 °C.
Refolding Assay	Antibacterial activity assay
Refolding Chaperones	None
Refolding Additives	cystamine
Additives Concentration	0.5 mM
Refolding Yield	13 mg
Purity	n/a
Notes	n/a

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