Refolding Record:
| Protein | |
|---|---|
| Protein Name | Prothrombin |
| Abbreviated Name | n/a |
| SCOP Family | Eukaryotic Proteases |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | P00735 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | Activation peptide fragment 2. |
| Chain Length | 426 |
| Molecular Weight | 48214.3 |
| Pi | 5.49 |
| Molecular Weight | 48214.3 |
| Disulphides | 4 |
| Full Sequence |
SGGSTTSQSPLLETCVPDRGREYRGRLAVTTSGSRCLAWSSEQAKALSKDQDFNPAVPLAENFCRNPDGDEEGAWCYVADQPGDFEYCDLNYCEEPVDGDLGDRLGEDPDPDAAIEGRTSEDHFQPFFNEKTFGAGEADCGLRPLFEKKQVQDQTEKELFESYIEGRIVEGQDAEVGLSPWQVMLFRKSPQELLCGASLISDRWVLTAAHCLLYPPWDKNFTVDDLLVRIGKHSRTRYERKVEKISMLDKIYIHPRYNWKENLDRDIALLKLKRPIELSDYIHPVCLPDKQTAAKLLHAGFKGRVTGWGNRRETWTTSVAEVQPSVLQVVNLPLVERPVCKASTRIRITDNMFCAGYKPGEGKRGDACEGDSGGPFVMKSPYNNRWYQMGIVSWGEGCDRDGKYGFYTHVFRLKKWIQKVIDRLGS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | DiBella EE, Maurer MC, Scheraga HA. (1995) Biologycal Chemistry, 270, 163-169 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | HMS174(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4-12 h |
| Expression Vector | plysS |
| Expression Protocol | Expression of Recombinant Prethrombin-2 E. coli HMS174(DE3)-pLys(S) harboring pThr-1 were grown at 37 °C in 5 ml of 2 YT (4% tryptone, 1% yeast extract, 10 mM NaCl, 1.4 mM KCl) containing 200 µg/ml ampicillin and 34 µg/ml chloramphenicol. The overnight cultures were diluted 1/200 into 1 liter of 2 YT containing 200 µg/ml ampicillin and 34 µg/ml chloramphenicol and grown at 37 °C with shaking. When the cells reached an OD = 0.4-1.0, expression from the T7 promoter was induced by adding isopropyl-1-thio--D-galactopyranoside to 0.4 mM, and growing was continued for another 4 to 12 h. The cells were then harvested by centrifugation at 7,800 rpm in a Beckman JA-10 rotor for 25 min at 4 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.4-1.0 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 ml of 0.5% Triton X-100, 2 mM EDTA, pH 8.0 |
| Solubilization Buffer | 7 M GdnHCl, 10 mM Tris, 1 mM EDTA, pH 8.0 |
| Refolding Buffer | 0.1 M NaHPO, 2 mM EDTA, 0.1% PEG, 0-4 M GdnHCl, 0.1 mM GSSG, 0.2 mM GSH, pH 7.4 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 7.4 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 0.2-0.1 mM |
| Refolding Protocol | Refolding of Recombinant Sulfonated Prethrombin-2 Lyophilized recombinant sulfonated prethrombin-2 was solubilized in 7 M GdnHCl, 10 mM Tris, 1 mM EDTA, pH 8.0, at a concentration of 4 mg/ml. The protein was diluted to a concentration of 25 µg/ml in folding buffer (0.1 M NaHPO, 2 mM EDTA, 0.1% PEG, 0-4 M GdnHCl, 0.1 mM GSSG, 0.2 mM GSH, pH 7.4). The solution was allowed to incubate for 24 h at room temperature and was then dialyzed, to remove GdnHCl, GSSG, and GSH at 4 °C, against 3 changes of 4 liters of 25 mM sodium phosphate, pH 7.4, containing 2 mM EDTA and 0.1% PEG. The refolded prethrombin-2 remained soluble upon removal of GdnHCl. The typical scale of a refolding experiment was 5 mg of sulfonated prethrombin-2 in 200 ml of folding buffer. The extent of prethrombin-2 refolding was assessed by a chromogenic assay. An aliquot of the dialyzed prethrombin-2 refolding solution (10-100 µl) in 975 µl of 25 mM sodium phosphate, pH 7.4, containing 0.15 M NaCl and 0.1% PEG, was activated to thrombin by adding 3 µl of E. carinatus snake venom (1 mg/ml) and incubating at 37 °C for 30 min. The snake venom was first pretreated with p-APMSF to inactivate contaminating serine proteases ()that interfere with measurements of S2238 activity(42) , and then desalted on a PD-10 column (Pharmacia) into 20 mM Tris, pH 8.0 buffer. The chromogenic substrate peptide S2238 (Kabi) at a final concentration of 0.1 mM was then added to the thrombin solution, and the absorbance at 405 nm was monitored at room temperature. All absorbance measurements were made on a modified Cary Model 14 spectrophotometer(43) .Purification of Refolded Recombinant Prethrombin-2 Refolded prethrombin-2 in 25 mM sodium phosphate, 2 mM EDTA, 0.1% PEG, pH 7.4, was purified on a 1-ml HiTrap heparin affinity column (Pharmacia) equilibrated with 25 mM sodium phosphate, pH 7.4. The sample was loaded at 1.2 ml/min and was eluted with a linear gradient from 0 M to 1 M NaCl in 25 mM sodium phosphate, pH 7.4. Recombinant prethrombin-2 was either activated immediately to thrombin or stored at -80 °C until needed. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 30-40 mg/l |
| Purity | n/a |
| Notes | n/a |