Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Isopenicillin N synthetase
Abbreviated Name	IPNS
SCOP Family	Unknown
Structure Notes
Organism	Flavobacterium sp.
UniProt Accession	P16020
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	327
Molecular Weight	36465.0
Pi	5.47
Molecular Weight	36465.0
Disulphides	Unknown
Full Sequence	MNRHADVPVIDISGLSGNDMDVKKDIAARIDRACRGSGFFYAANHGVDLAALQKFTTDWHMAMSAEEKWELAIRAYNPANPRNRNGYYMAVEGKKANESFCYLNPSFDADHATIKAGLPSHEVNIWPDEARHPGMRRFYEAYFSDVFDVAAVILRGFAIALGREESFFERHFSMDDTLSAVSLIRYPFLENYPPLKLGPDGEKLSFEHHQDVSLITVLYQTAIPNLQVETAEGYLDIPVSDEHFLVNCGTYMAHITNGYYPAPVHRVKYINAERLSIPFFANLSHASAIDPFAPPPYAPPGGNPTVSYGDYLQHGLLDLIRANGQT
Notes	n/a

Expression
Report	Landman O, Shiffman D, Av-Gay Y, Aharonowitz Y, Cohen G. (1991) FEMS Microbiology Letters, 68, 239-44
Project Aim	Over expression & Renaturation
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	2 h
Expression Vector	pET11d
Expression Protocol	E. coli B (DE3)/lysS strains containing IPNS recombinant plasmids were grown at 37°C in LB medium supplemented with kanamycin (50 ug/ml) and chloramphenicol (25 ug/ml) to OD650 0.6. Synthesis of IPNS was performed by addition of the inducer isopropylthio-/3-D-galactoside (IPTG) to the culture to a final concentration of 0.4 mM and incubating for 2 h. Aliquots of the cultures were sonicated and the crude extracts were assayed for IPNS protein and activity directly (see below) or after centrifugation for 5 min in a minifuge to separate the insoluble IPNS pellet fraction from the soluble supernatant fraction.
Method of Induction	IPTG
Cell Density at Induction	OD 0.6 = 650
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	not specified
Solubility	partial

Refolding
Refolding Method	Column refolding: Size-exclusion chromatography
Wash Buffer	50 mM Tris. HC1 (pH 8.0), 1 mM dithiothreitol (DTT), 0.01 mM ethylene di aminotetraacetic acid (EDTA) and 10% glycerol (0.5 ml)
Solubilization Buffer	50 mM Tris. HC1 (pH 8.0), 0.1 M DTT, 5 M urea, 1 mM EDTA
Refolding Buffer	50 mM Tris. HC1 (pH 8.0), 1 mM DTT and 0.01 mM EDTA.
Pre-Refolding Purification	not specified
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	DTT
Redox Agent Concentration	1 mM
Refolding Protocol	Cultures of IPTG-treated cells (500 ml) were chilled, concentrated 100-fold by centrifugation and sonicated in a Branson sonifier equipped with a microtip. The crude extract (5 ml) was spun for 10 rain at 10000 × g and the pellet was resuspended in 50 mM Tris. HC1 (pH 8.0), 1 mM dithiothreitol (DTT), 0.01 mM ethylene diaminotetraacetic acid (EDTA) and 10% glycerol (0.5 ml). Sonication was repeated five times with intermittent cooling of the sample on ice and the final pellet was stored at -70°C. IPNS was denatured and solubilized by adjusting the preparation to 5 ml containing 50 mM Tris. HC1 (pH 8.0), 0.1 M DTT, 5 M urea, 1 mM EDTA and incubating at room temperature for 2 h. Urea was removed and active IPNS was recovered by passing the denatured material through a G-25 Sephadex column (Pharmacia) previously equilibrated with 50 mM Tris. HC1 (pH 8.0), 1 mM DTT and 0.01 mM EDTA. This process we refer to as refolding; it is not meant to imply that the active IPNS recovered is identical to the native protein. Further purification was carried out by chromatography on DEAE-Sephacel (Pharmacia) in the above buffer employing a gradient of 50-500 mM Tris. HC1 (pH 8.0).
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	30%
Purity	n/a
Notes	n/a

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