Refolding Record:
| Protein | |
|---|---|
| Protein Name | Arginine Deiminase |
| Abbreviated Name | AD |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | listeria monocytogenes |
| UniProt Accession | Q8YAS0 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 411 |
| Molecular Weight | 47114.9 |
| Pi | 5.11579 |
| Molecular Weight | 47114.9 |
| Disulphides | Unknown |
| Full Sequence |
MKMEQALNITSEIGKLQTVLVKRPGSELENITPEYLESLLFDDIPYLKMMQKEHDFFAKTMRDSNIEVLYLEKLAAEALR
EANNKESFLTKMIKESNQMDESALYVRDYLMSFDEEEMIRKLMSGLKKSEIPERKKKHLNEMMDEQYPFFLDPLPNLYFT
RDPAAVIGNGVTINRMFQPARRRESIFIELILKHHPRFSNQDIPLWSGRGEPFSLEGGDELVLNEETVLVGVSERTDARA
VERLAESLFNRSPKIKRVLAVEIPETRSFMHLDTVFTMVNFAQFTIHPAIQNQQGELNIYILEKSENGLEITPRRDFQRV
IAEVLDEPEIDFIPCGGEDVIVSAREQWNDGANTLAIAPGEVITYDRNQVSNDLLRSAGIKVHEVISSELSRGRGGPRCM
TMPLVRENLK.
|
| Notes | The target protein was inserted into plasmid pET30a and then transformed E.coli(rosetta). |
| Expression | |
|---|---|
| Report | Unpublished (0) J Virol Methods, 0, 0 |
| Project Aim | Analysis |
| Fusion | C-terminal hexahis + pro seq |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | E.coli |
| Expression Strain | rosetta |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pet30a |
| Expression Protocol | E. coli £¨rosetta)cells harboring pET30a-arcA were incubated in a 1L shake flask containing 200 ml LB medium supplemented with 30 lg/ml Kanamycin at 37C. At 0.6 OD600, IPTG was added to a final concentration of 0.4 mM to induce the expression of arginine deiminase. After 3 h of induction, the cell pellets were collected (6,000g, 10 min at 4C), washed twice with PBS. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 530 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Chemicals |
| Pre-Refolding Purification | Affinity chromotography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 10mM potassium phosphate buffer(pH7.0) containaing 4%(v/v) TritonX-100 and 1 mM EDTA. |
| Solubilization Buffer | 6M Gdn-HCl |
| Refolding Buffer | 10mM potassium phosphate buffer(pH7.0) |
| Pre-Refolding Purification | Affinity chromotography |
| Tag Cleaved | no |
| Refolding pH | 7.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 45h |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a |
| Refolding Protocol | 1¡¢ Add the solubilized, unfolded protein (in denaturant) to 20-100x volume refolding buffer. 2¡¢ Leave the refolding mixture for at least 45 hours at4oC with gentle stirring. 3. Centrifuge or filter refolded protein mixture to remove any insoluble material that may have formed. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | DTT |
| Refolding Yield | no activity |
| Purity | n/a |
| Notes | n/a |