Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Lipase
Abbreviated Name	lip4
SCOP Family	Fungal lipases
Structure Notes
Organism	Pseudomonas fluorescens
UniProt Accession	Q4KJ24
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	296
Molecular Weight	32427.0
Pi	9.89
Molecular Weight	32427.0
Disulphides	Unknown
Full Sequence	MSQELATRYPLVLVPGMLGFVRLLLYPYWYGIIPALRRGGAQVIAVQVSPLNSSEVRGEQLLAQIQRIMAETGAARVNLIGHSQGALTARYAAARRPDWVASVTSVAGPNHGSELADYLQRHSPAHSLRGRVLSLLLRGISSLMRLLETGYRGPKQPVDIHASHQSLTTEGVALFNRQYPQGLPQEWGGQGPAQVDGVHYYSWSGILQPGKTNRGRNLFDGTNRSCRLFARTFVREAGQCDGMVGRYSSHLGQVIGDDYPLDHFDIVNQSLGLVGKGAEPIRLFVEHARRLKAAGL
Notes	n/a

Expression
Report	Kim KR, Kwon DY, Yoon SH, Kim WY, Kim KH. (2005) Protein Expression and Purification, 39, 124-129
Project Aim	Purification & characterization
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21
Expression Temp	0.0
Expression Time	00
Expression Vector	pTTY2
Expression Protocol	Bacterial cells expressing PFL were grown according to a previously described method [2] and [11]. The cultured cells (typically 20 g wet weight) were harvested by centrifugation (15 min at 8000g), resuspended in 30 ml of lysis buffer (50 mM Tris–HCl, pH 8.0, and 1 mM EDTA), and lysed by sonication for 1 min in the presence of lysozyme (0.2 mg/ml). Inclusion bodies were separated by centrifugation (30 min 12,000g), and pellets were washed with lysis buffer containing 2% Triton X-100, to remove cell debris and other impurities.
Method of Induction	Not Stated
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	Column refolding: Size-exclusion chromatography
Wash Buffer	50 mM Tris–HCl, pH 8.0, and 1 mM EDTA, 2% Triton X-100
Solubilization Buffer	50 mM Tris–HCl, pH 8.0, 8 M urea, and 20 mM β-mercaptoethanol
Refolding Buffer	20 mM Tris–HCl, pH 8.0, and 10 mM CaCl2
Pre-Refolding Purification	not specified
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Inclusion bodies were separated by centrifugation (30 min 12,000g), and pellets were washed with lysis buffer containing 2% Triton X-100, to remove cell debris and other impurities. Pellets were then resuspended in 10 ml denaturation buffer (50 mM Tris–HCl, pH 8.0, 8 M urea, and 20 mM β-mercaptoethanol), insoluble components were removed by centrifugation (30 min 12,000g), and the supernatant was filtered using a 0.45 μm syringe filter. SEPROS was carried out at 4 °C using refolding buffer (20 mM Tris–HCl, pH 8.0, and 10 mM CaCl2) and a Sephacryl S-200 on Biologic LP system. The solubilized inclusion bodies were applied to the column (2.5 × 75 cm) in 8 M urea (1.5 ml), equilibrated with the refolding buffer, and eluted at 1.5 ml/min. The active fractions from SEPROS were pooled, concentrated, and applied to gel filtration FPLC using a Superdex 200 HR prepacked column. The active fractions were further separated by ion exchange FPLC using a Mono Q prepacked column, and fractions with lipase activity were pooled, concentrated, and subjected to a second gel filtration FPLC using Superdex 200. PFL was also refolded using SEPROS and further purified in the absence of Ca2+, which was used as a control.
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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