Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Serpin A5 (also known as Protein Inhibitor 3, Plasminogen Activator Inhibitor 3)
Abbreviated Name	PLn
SCOP Family	Serpins
Structure Notes
Organism	Human
UniProt Accession	P05154
SCOP Unique ID	n/a
Structure Solved
Class	Multi-domain proteins (alpha and beta)
Molecularity	Unknown

Construct
Full Length	n
Domain	Recombinant N-terminal domain+kringle 1
Chimera	n/a
Variants	n/a
Chain Length	77
Molecular Weight	8579.0
Pi	9.77
Molecular Weight	8579.0
Disulphides	Unknown
Full Sequence	MQLFLLLCLVLLSPQGASLHRHHPREMKKRVEDLHVGATVAPSSRRDFTFDLYRALASAAPSQNIFFSPVSISMSLA
Notes	n/a

Expression
Report	Douglas JT, von Haller PD, Gehrmann M, Llinás M, Schaller J. (2002) Biochemistry, 41, 3302-3310
Project Aim	Folding
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	5.5 h
Expression Vector	PQE8
Expression Protocol	Expression and Isolation of rNK1. The protocol followed is essentially that of Cleary et al. (18) and Marti et al. (19). Cells were grown at 37 C in 2 × YT medium (100 g of ampicillin/mL, 25 g of kanamycin/mL) in 2 L round-bottom flasks to an OD600 of about 0.7-0.9. To induce the production of the recombinant proteins, isopropyl-thio--D-galactopyranoside was added to a final concentration of 2 mM. Cells were grown for an additional 5.5 h at 37 C and harvested by centrifugation for 30 min (4000g, 4 C). The cell paste was stored at -20 C. The protein extracts were analyzed on 10% SDS-PAGE gels and blotted onto nitrocellulose membranes. Novel bands were detected by Ponceau S staining and were verified as domains of Pgn by an ELISA based on polyclonal antibodies against native Pgn.
Method of Induction	IPTG
Cell Density at Induction	OD 0.7-0.9 = 600
Cell Disruption Method	Not stated
Lytic Agent	None
Pre-Refolding Purification	RP-HPLC
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	6 M guanidine hydrochloride in 0.1 M sodium phosphate, pH 8; 5 mL/g of cell paste
Solubilization Buffer	6 M guanidine hydrochloride in 0.1 M sodium phosphate, pH 5
Refolding Buffer	5 mM 1,4 dithio-dl-threitol, 50 mM sodium phosphate buffer, pH 8, containing 1.25 mM each of reduced and oxidized glutathione
Pre-Refolding Purification	RP-HPLC
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	6 h
Redox Agent	GSH/GSSG/DTT
Redox Agent Concentration	1.25/1.25/5 mM
Refolding Protocol	Refolding and Purification of rNK1. During refolding, gentle dialysis was performed to minimize partial precipitation of the protein. From earlier studies (12, 19, 20), it is known that under these conditions the disulfide bonds of kringle domains and of the N-domain pair correctly due to the strong conformational determinants which seem to be present in both structures. The pH of the effluent was adjusted to 8, and 1,4 dithio-dl-threitol was added to a final concentration of 5 mM. After being stirred overnight at 4 C, the solution was slowly (6 h) added, with continuous stirring at 4 C, to 4 volumes of 50 mM sodium phosphate buffer, pH 8, containing 1.25 mM each of reduced and oxidized glutathione. The solution was dialyzed against 50 mM sodium phosphate, pH 8, and loaded onto a column of lysine-substituted Bio-Gel P-300 (lysine-Bio-Gel, 1.5 × 5 cm). After the column was washed with loading buffer, rNK1 was eluted with 50 mM sodium phosphate, pH 8, containing 200 mM 6-AHA (Figure 2B), dialyzed against water, acidified to pH 3-4 with formic acid, and lyophilized. Refolding was monitored by RP-HPLC on an Aquapore Butyl column. The totally reduced form of rNK1 eluted at 45% B as a sharp, single peak, and the completely refolded form eluted at about 40% B while the folding intermediates appeared between. This shift might be caused by the change of the hydrophobicity on the surface of the proteins. The finally purified rNK1 eluted as a sharp, symmetrical peak upon RP-HPLC on an Aquapore Butyl column (Figure 2C). The N-terminal sequence Met-Arg-Gly-Ser-His was checked by automated Edman degradation, and the amino acid composition yielded values compatible with the protein sequence and confirmed the absence of 6-AHA.
Refolding Assay	Far-UV Circular Dichroism
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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