Refolding Record:
| Protein | |
|---|---|
| Protein Name | Tissue factor pathway inhibitor |
| Abbreviated Name | TFPI |
| SCOP Family | Small Kunitz-type inhibitors & BPTI-like toxins |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P10646 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Kunitz 2 domain |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 64 |
| Molecular Weight | 7464.3 |
| Pi | 4.53292 |
| Molecular Weight | 7464.3 |
| Disulphides | 9 |
| Full Sequence |
MAPDFCFLEE DPGICRGYIT RYFYNNQTKQ CERFKYGGCL GNMNNFETLE ECKNICEDGP NGFQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Day KC and Welsch DJ (1992) Thrombosis Research, 68, 369-381 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 30.0 |
| Expression Time | n/a |
| Expression Vector | pMON5842 |
| Expression Protocol | 10L fermentations were performed using M9 media containing 2% casamino acids with glucose added to a concentration of approximately 2.5g/L. Fermentation temperature was maintained at 30degC, the pH was controlled at 7.0 with ammonium hydroxide. The culture was grown until OD550 reached 55, at which point expression was induced with 0.25mM IPTG. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 550 = 55 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | not stated |
| Solubilization Buffer | 50mM Tris pH 8.0, 100mM NaCl, 7.5M Urea |
| Refolding Buffer | 50mM Tris pH 8.5, 100mM NaCl |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The wet pellet of 3L of broth was suspended in 300ml of 10mM EDTA pH 8.0 and lysed via sonication. The insoluble pellet was collected by centrifugation, then washed three times prior to being slurried into 50mL solubilization buffer. DTT was added to 200mM and the solution was incubated at 55degC for 30min. The solution was centrifuged again and the supernatant filtered. The filtered protein was then diluted ten-fold into solubilization buffer, then dialyzed exhaustively against refolding buffer. The oxidized protein was lyophilized and resuspended in 25mM ammonium bicarbonate pH 8.0, then purified using gel filtration (Superdex 75). The pooled monomer was then further purified by reverse-phase HPLC. |
| Refolding Assay | Amino acid sequencing |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | See Obukowicz et al. (1990) Biochemistry, v.29, 9737-9745 for details of expression protocol |