Refolding Record:
| Protein | |
|---|---|
| Protein Name | Fibritin |
| Abbreviated Name | Fibritin |
| SCOP Family | Fibritin |
| Structure Notes | |
| Organism | Bacteriophage T4 |
| UniProt Accession | P10104 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Trimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | Fibritin Mutant XN, composed of residues 1-457 |
| Chain Length | 477 |
| Molecular Weight | 50481.9 |
| Pi | 4.57 |
| Molecular Weight | 50481.9 |
| Disulphides | 0 |
| Full Sequence |
TDIVLNDLPFVDGPPAEGQSRISWIKNGEEILGADTQYGSEGSMNRPTVSVLRNVEVLDKNIGILKTSLETANSDIKTIQGILDVSGDIEALAQIGINKKDISDLKTLTSEHTEILNGTNNTVDSILADIGPFNAEANSVYRTIRNDLLWIKRELGQYTGQDINGLPVVGNPSSGMKHRIINNTDVITSQGIRLSELETKFIESDVGSLTIEVGNLREELGPKPPSFSQNVYSRLNEIDTKQTTVESDISAIKTSIGYPGNNSIITSVNTNTDNIASINLELNQSGGIKQRLTVIETSIGSDDIPSSIKGQIKDNTTSIESLNGIVGENTSSGLRANVSWLNQIVGTDSSGGQPSPPGSLLNRVSTIETSVSGLNNAVQNLQVEIGNNSAGIKGQVVALNTLVNGTNPNGSTVEERGLTNSIKANETNIASVTQEVNTAKGNISSLQGDVQALQEAGDPAANKARKEAELAAATAEQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Boudko, S. P., Londer, Y. Y., Leetarov, A. V., Sernova, N. V., Engel, J., Mesyanzhinov, V. V. (2002) Eur J Biochem., 269, 833-841 |
| Project Aim | Folding,Structural Studies |
| Fusion | C-terminal sequence |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pET19b |
| Expression Protocol | Cells grown in 500ml of 2x tryptone/yeast medium. After induction cells were incubated with vigorous aeration and harvested by centrifugation(6000g, 10min, 4degC). Cells were then resuspended in 10ml of buffer A (50mM TrisHCl, pH 8.0, 1 mM EDTA) and sonicated. Cell debris was removed via centrifugation(3500g for 30 min) and the supernatant was removed. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M urea |
| Refolding Buffer | 50mM TrisHCl, pH 8.0, 2mM EDTA, 2mM PMSF |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 3-4days |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pellet, containing inclusion bodies, was then resuspended in 0.5 ml solubilization buffer for 10min and centrifuged(10,000g for 30min). The supernatant was then mixed with 50ml of refolding buffer and incubated before concentration to 2ml. The protein solution was purified on an hydroxylapatite column equilibrated with 10mM Na phosphate, pH 8.0). The column was then washed with 10mM Na phosphate and flow through fractions that contained protein were dialyzed against buffer A |
| Refolding Assay | Far-UV Circular Dichroism |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 15% |
| Purity | n/a |
| Notes | n/a |