Refolding Record:
| Protein | |
|---|---|
| Protein Name | EnterotoxinA C-terminal domain |
| Abbreviated Name | TcdA |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Clostridium difficile |
| UniProt Accession | P16154 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | C-terminal domain with a 607aa intrachain deletion, aa.1753-2681 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 332 |
| Molecular Weight | 38143.6 |
| Pi | 8.89703 |
| Molecular Weight | 38143.6 |
| Disulphides | Unknown |
| Full Sequence |
MRGSEESNKKIL QKIRIKGILS NTQSFNKMSI DFKDIKKLSL GYIMSNFKSF NSENELDRDH LGFKIIDNKT YYYDEASKLV KGLININNSL FYFDPIESNL VTGWQTINGK KYYFDINTGA ASTSYKIING KHFYFNNNGV MQLGVFKGPD GFEYFAPANT QNNNIEGQAI VYQSKFLTLN GKKYYFDNDS KAVTGW Q TIDGKKYYFN LNTAEAATGW QTIDGKKYYF NTNTSIASTG YTIINGK HFYFNTDGIM QIGVFKGPDG FEYFAPANTD ANNIEGQAIR YQNRFLYLHD NIYYFGNNS KAVTGWQTIN GNVYYFMPDT A HHHHHH
|
| Notes | See UniProt ID: O32351_CLODI for sequence used in this study |
| Expression | |
|---|---|
| Report | Letourneur O, Ottone S, Delauzun V, Bastide MC, Foussadier A. (2003) Protein Expression and Purification, 31, 276-285 |
| Project Aim | Undefined |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pMR |
| Expression Protocol | A fresh overnight culture of cells was diluted to 1:25 in 1L of 2TY medium containing 2% glucose and 100microgram/ml ampicillin. Cells were grown at 37degC with stirring (250rmp) until OD600 reached 0.7-0.9. The culture was then induced with 1mM IPTG for 4h at 37degC. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD 600 = 0.7-0.9 |
| Cell Disruption Method | Chemical |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 40mM sodium bicarbonate, 300mM NaCl, 1%SDS, 20mM beta-mercaptoethanol, pH 9.6 |
| Refolding Buffer | PBS, 8mM imidazole |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The harvested cell pellet was resuspended in 100ml PBS, 1mM MgCl (pH 7.4) containing 1mg/ml lysozyme, 2.5U/ml benzonase and protease inhibitor cocktail without EDTA. After incuabtion for 1h at reoom temperature with stirring, the lysate was centrifuged (30min x 10000g at 4degC). The pelleted inclusion bodies were then resuspended in solubilization buffer and incubated for 1h at 37degC with stirring. Following stirring, the solution was diluted to 1/4 with PBS containing 8mM imidazole (pH 8.0) and centrifuged (30min x 10000g at 20degC). The supernatant was then loaded onto a 10ml Ni-NTA column equilibrated in 2xPBS, 0.25% SDS, 5mM beta-mercaptoethanol and 6mM imidazole, pH 8.0 at 1ml/h and room temperature. The protein was then eluted with 2xPBS, 0.25% SDS, 5mM beta-mercaptoethanol and 100mM imidazole, pH 7.5. |
| Refolding Assay | Immunoassay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 89mg/L culture |
| Purity | |
| Notes | Similar protocol for cytotoxin B (TcdB) (record 1480), with some variations |