Refolding Record:
| Protein | |
|---|---|
| Protein Name | Annexin 5A |
| Abbreviated Name | ANV |
| SCOP Family | Annexin |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P08758 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | ANV-Tissue factor pathway inhibitor Kunitz domains 1 and 2, aa.22-161 |
| Variants | C316A |
| Chain Length | 461 |
| Molecular Weight | 52292.1 |
| Pi | 5.01 |
| Molecular Weight | 52292.1 |
| Disulphides | 8 |
| Full Sequence |
GSAQVLRGTVTD FPGFDERADA ETLRKAMKGL GTDEESILTL LTSRSNAQRQ EISAAFKTLF GRDLLDDLKS ELTGKFEKLI VALMKPSRLY DAYELKHALK GAGTNEKVLT EIIASRTPEE LRAIKQVYEE EYGSSLEDDV VGDTSGYYQR MLVVLLQANR
DPDAGIDEAQ VEQDAQALFQ AGELKWGTDE EKFITIFGTR SVSHLRKVFD KYMTISGFQI EETIDRETSG NLEQLLLAVV KSIRSIPAYL AETLYYAMKG AGTDDHTLIR VMVSRSEIDL FNIRKEFRKN FATSLYSMIK GDTSGDYKKA LLLLAGEDD
MHSFCAFKADD GPCKAIMKRF FFNIFTRQCE EFIYGGCEGN QNRFESLEEC KKMCTRDNAN RIIKTTLQQE KPDFCFLEED PGICRGYITR YFYNNQTKQC ERFKYGGCLG NMNNFETLEE CKNICEDGPN GFQVDNYGT
|
| Notes | Tissue factor pathway inhibitor (TFPI) UniProt ID: TFPI_HUMAN, fragment used contains Kunitz domains 1 and 2 (residues 22-161) |
| Expression | |
|---|---|
| Report | Chen HH, Vicente CP, He L, Tollefsen DM, Wun TC (2005) Blood, 105, 3902-3909 |
| Project Aim | Drug Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pET20b+ |
| Expression Protocol | 1L medium was inoculated with 10ml overnight culture and grown at 37degC until OD600reached 0.5. Expression was induced with 1mM IPTG and incubation continued for 4h before cells were harvested by centifugation. The cell pellet was stored at -80degC. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5 |
| 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 using a Q-sepharose column followed by binding and elution from anionic liposomes |
| Refolding Assay | Bioactivity,Inhibitory activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |