Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Prostasin
Abbreviated Name	PRSS8
SCOP Family	Unknown
Structure Notes
Organism	Human
UniProt Accession	Q16651
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Heterodimer

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	VAriant 26
Chain Length	312
Molecular Weight	33337.8
Pi	5.39
Molecular Weight	33337.8
Disulphides	5
Full Sequence	AEAPCGVAPQARITGGSSAVAGQWPWQVSITYEGVHVCGGSLVSEQWVLSAAHCFPSEHHKEAYEVKLGAHQLDSYSEDAKVSTLKDIIPHPSYLQEGSQGDIALLQLSRPITFSRYIRPICLPAANASFPNGLHCTVTGWGHVAPSVSLLTPKPLQQLEVPLISRETCNCLYNIDAKPEEPHFVQEDMVCAGYVEGGKDACQGDSGGPLSCPVEGLWYLTGIVSWGDACGARNRPGVYTLASSYASWIQSKVTELQPRVVPQTQESQPDSNLCGSHLAFSSAPAQGLLRPILFLPLGLALGLLSPWLSEH
Notes	n/a

Expression
Report	Rickert KW, Kelley P, Byrne NJ, Diehl RE, Hall DL, Montalvo AM, Reid JC, Shipman JM, Thomas BW, Munshi SK, Darke PL, Su HP. (2008) J Biol Chem, 1, 1
Project Aim	Structural Studies
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	E coli
Expression Strain	BL21(DE3)
Expression Temp	1.0
Expression Time	1
Expression Vector	n/a
Expression Protocol	Bacterial expression constructs contained the original native propeptide sequence at the N-terminus, but with the addition of an enterokinase recognition sequence to allow specific cleavage to generate the active enzyme. Detailed methods for the expression of prostasin in both baculovirus and E. coli are provided in the Supplementary Experimental Procedures
Method of Induction	Not Stated
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Not stated
Lytic Agent	None
Pre-Refolding Purification	Ni-NTA agrose chromatography
Solubility	insoluble

Refolding
Refolding Method	Column refolding: Nickel-chelating chromatography
Wash Buffer	n/a
Solubilization Buffer	8 M urea with 0.1 M Tris/HCl, pH 8.0, and 2 mM DTT
Refolding Buffer	1 M L-arginine, 0.1 M Tris/HCl, 5 mM reduced glutathione, and 0.5 mM oxidized glutathione, pH 8.0
Pre-Refolding Purification	Ni-NTA agrose chromatography
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	1.0
Protein Concentration	10.0 mg/L
Refolding Time	n/a
Redox Agent	GSH/GSSG
Redox Agent Concentration	5/0.5 mM
Refolding Protocol	Prostasin variants 26 and 28 (Figure 1) were solubilized from inclusion bodies in 8 M urea with 0.1 M Tris/HCl, pH 8.0, and 2 mM DTT after extensive washing. Solubilized protein was bound to Ni-NTA Superflow resin (Qiagen) and eluted with 0.3 M imidazole. The urea-solubilized material was then refolded in a buffer containing 1 M L-arginine, 0.1 M Tris/HCl, 5 mM reduced glutathione, and 0.5 mM oxidized glutathione, pH 8.0, at a final concentration of 10 mg/L. Following diafiltration, protein was purified via Ni(II) affinity and anion exchange chromatography. Eluted fractions were evaluated via SDS-PAGE and mass spectrometry (MS), and fractions in which prostasin was detected by MS were then pooled for further processing. Prostasin zymogen was converted to the active form by addition of enterokinase (EKMax, Invitrogen) at a final concentration of 2 U/mL (7.5 U/mg prostasin) in the presence of 0.5 mM reduced glutathione. The resultant cleavage reaction was maintained at 4ºC for 48 hours and monitored by MS and SDS-PAGE. Following completion of cleavage as determined by MS, the reaction was incubated at 4ºC overnight after the addition of 1 mM oxidized glutathione. Active prostasin was purified from enterokinase and misfolded prostasin via Ni(II) affinity and anion exchange chromatography.
Refolding Assay	SDS-PAGE,enzyme activity
Refolding Chaperones	None
Refolding Additives	L-Arginine
Additives Concentration	1 M
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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