Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Pancreatic secretory trypsin inhibitor
Abbreviated Name	PSTI-I
SCOP Family	Unknown
Structure Notes
Organism	Rat
UniProt Accession	P09655
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	61
Molecular Weight	6658.6
Pi	8.65
Molecular Weight	6658.6
Disulphides	Unknown
Full Sequence	GNPPAEVNGKTPNCPKQIMGCPRIYDPVCGTNGITYPSECSLCFENRKFGTSIHIQRRGTC
Notes	n/a

Expression
Report	Graf R, Klauser S, Fukuoka SI, Schiesser M, Bimmler D. (2003) Pancreatology., 3, 195-206
Project Aim	Analysis
Fusion	None
Protein Expression and Production	Protein synthesized by chemical means (not recombinant) and refolded.
Expression Host	None
Expression Strain	None
Expression Temp	0.0
Expression Time	0
Expression Vector
Expression Protocol
Method of Induction	Not Stated
Cell Density at Induction	OD =
Cell Disruption Method	None
Lytic Agent	None
Pre-Refolding Purification	not specified
Solubility

Refolding
Refolding Method	Dilution
Wash Buffer	n/a
Solubilization Buffer	8 M urea, 0.3 M sodium sulfite pH 8.0
Refolding Buffer	25 mM Tris-HCl pH 8.0, 5 mM EDTA, 8 mM reduced glutathione, 4 mM oxidized glutathione
Pre-Refolding Purification	not specified
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	1 day
Redox Agent	GSH/GSSG
Redox Agent Concentration	8/4 mM
Refolding Protocol	Renaturation of PSTI-I, Renaturation of PSTI-I followed a two-step procedure by sulfonation of the peptide and subsequent slow equilibration with oxidizing and reducing agents. To sulfonate the peptide, the reagent 2-nitro-5-thiosulfobenzoate (NTSB) was synthesized from 5,5)-dithiobis-2- nitrobenzoic acid (Aldrich) [9]. One milligram PSTI-I was completely denatured by dissolving it in 1 ml 8 M urea, 0.3 M sodium sulfite pH 8.0, and stirred for 30 min at room temperature. NTSB (125 Ìl) was added and the solution was stirred for another 30 min. The reaction product was purified over a P-10 BioRad size exclusion column (1.6 ! 20 cm) equilibrated with 25 mM Tris pH 8.0, 5 M urea, 6 mM EDTA [10]. To identify the peptide, eluates were diluted 1:250 in carbonate buffer and a 96-well plate was coated overnight. The presence of the peptide was detected with the ELISA procedure described below. To the combined positive fractions, 2.5 vol of the oxidizing/ reducing solution (25 mM Tris-HCl pH 8.0, 5 mM EDTA, 8 mM reduced glutathione, 4 mM oxidized glutathione, all Sigma) were added slowly over a period of 2 h [10, 11]. The reaction was allowed to equilibrate for 1 day at room temperature, and was then dialyzed extensively against 10 mM Tris pH 8.0, 0.1 M NaCl. The peptide was purified again by RP-HPLC as described above. The oxidized product was identified by mass spectrometry (calculated molecular weight: 6,652) and had the same value as that reported by Lin et al. [12] who also chemically synthesized PSTI-I.
Refolding Assay	Binding assay,Statistical Analysis
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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