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 | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 278 |
| Molecular Weight | 32152.6 |
| Pi | 8.46 |
| Molecular Weight | 32152.6 |
| Disulphides | >10 |
| Full Sequence |
MADS EEDEEHTIIT DTELPPLKLM HSFCAFKADD GPCKAIMKRF FFNIFTRQCE EFIYGGCEGN QNRFESLEEC KKMCTRDNAN RIIKTTLQQE KPDFCFLEED PGICRGYITR YFYNNQTKQC ERFKYGGCLG NMNNFETLEE CKNICEDGPN GFQVDNYGTQ LNAVNNSLTP QSTKVPSLFE FHGPSWCLTP ADRGLCRANE NRFYYNSVIG KCRPFKYSGC GGNENNFTSK QECLRACKKG FIQRISKGGL IKTKRKRKKQ RVKIAYEEIF VKNM
|
| Notes | A single extra Ala residue at start of sequence, remaining from signal sequence - wild-type mature sequence starts at DSEEDEE |
| Expression | |
|---|---|
| Report | Diaz-Collier JA, Palmier MO, Kretzmer KK, Bishop BF, Combs RG, Obukowicz MG, Fraier RB, Bild GS, Joy WD, Hill SR, Duffin KL, Gustafson ME, Junger KD, Grabner RW, Galluppi GR, Wun T (1994) Thrombosis and Haemostasis, 3, 339-46 |
| Project Aim | Recombinant Protein Expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | MON105 |
| Expression Temp | 30.0 |
| Expression Time | 4h |
| Expression Vector | pMON5557 |
| Expression Protocol | Cells were grown in 10L of M9 minimal salts media supplemented with 20g/l casamino acids at 37degC, 1000rpm, air flow rate of 15L/min, 10psi backpressure, with pH controlled at 7.0 with ammonium hydroxide. Residual glucose concentration was controled at 1.0g/l. When OD550 reached 46, the temperature was shifted from 37degC to 30degC and 1.0mM IPTG was added. The culture was harvested for 4h, then cells were harvested by concentration of the media followed by centrifugation. the cell paste was frozen at -80degC. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 550 = 46 |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 50mM TrisHCl pH 8, 7.5M urea |
| Refolding Buffer | 10mM TrisHCl pH 8, 3M urea, 0.005% Brij 35, 0.15M NaCl, 2mM L-cysteine |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 8 days |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 2mM |
| Refolding Protocol | Frozen cell paste was resuspended in cold Milli-Q water at 75g/l. The cells were thoroughly dispersed with a homogenizer for 30min on ice, then lysed by three passes through a high-pressure homogenizer at 12000psi. The lysate was centrifuged (16270g, 20min), and the inclusion body pellets were resuspended in 1L of cold MilliQ water and dispersed by homogenization again. The inclusion bodies were then passed through the high pressure homogenizer two more times then collected by centrifugation and stored at -80degC. 1g of inclusion bodies was dispersed in 40ml solubilization buffer by homogenization and vortexing. After the protein was dissolved, 800mg of sodium sulfite was added and the mixture was shaken at room temperature for 30min. Then 400mg of sodium dithionite was added and the mixture was shaken at 4degC overnight. The solution was was dialyzed agast 400ml of 20mM TrisHCl pH 8, 4M urea for more than 5h at 4degC. The solution was centrifuged (48400g, 1h) and filtered then stored at -80degC. The protein was then fractionated on a MonoQ HR5/5 column in 20mM TrisHCl pH 8, 6M urea, 0.01% Brij35, with a gradient of NaCl from 0.15-0.4M. The eluted protein was then diluted to A280=0.07 in the same buffer containing 0.3M NaCl, solid L-cysteine was added to 2mM and the solution was incubated at room temperature for 24h then diluted 1:1 with water. 1mM L-cysteine was added, the solution was incubated at room tmperature for another 24h and then incubated at 4degC for up to 8 days. The protein was then purified further by passage down a Mono S Hr5/5 column. |
| Refolding Assay | Mass spectrometry,Inhibitory activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 68% |
| Purity | n/a |
| Notes | n/a |