Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cytotoxin B C-terminal domain |
| Abbreviated Name | TcdB |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Clostridium difficile |
| UniProt Accession | P18177 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | C-terminal domain, aa.1755-2360 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 614 |
| Molecular Weight | 70851.7 |
| Pi | 4.21964 |
| Molecular Weight | 70851.7 |
| Disulphides | Unknown |
| Full Sequence |
MREENKVS QVKIRFVNVF KDKTLANKLS FNFSDKQDVP VSEIILSFTP SYYEDGLIGY DLGLVSLYNE KFYINNFGMM VSGLIYINDS LYYFKPPVNN LITGFVTVGD DKYYFNPING GAASIGETII DDKNYYFNQS GVLQTGVFST EDGFKYFAPA NTLDENLEGE AIDFTGKLII DENIYYFDDN YRGAVEWKEL
DGEMHYFSPE TGKAFKGLNQ IGDYKYYFNS DGVMQKGFVS INDNKHYFDD SGVMKVGYTE IDGKHFYFAE NGEMQIGVFN TEDGFKYFAH HNEDLGNEEG EEISYSGILN FNNKIYYFDD SFTAVVGWKD LEDGSKYYFD EDTAEAYIGL SLINDGQYYF NDDGIMQVGF VTINDKVFYF SDSGIIESGV QNIDDNYFYI DDNGIVQIGV FDTSDGYKYF APANTVNDNI YGQAVEYSGL VRVGEDVYYF GETYTIETGW IYDMENESDK YYFNPETKKA CKGINLIDDI KYYFDEKGIM RTGLISFENN NYYFNENGEM QFGYINIEDK MFYFGEDGVM QIGVFNTPDG FKYFAHQNTL DENFEGESIN YTGWLDLDEK RYYFTDEYIA ATGSVIIDGE
EYYFDPDTAQ HHHHHH
|
| Notes | n/a |
| 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 | IPTG |
| Cell Density at Induction | OD 600 = 0.7-0.9 |
| Cell Disruption Method | Chemical |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | partial |
| 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 pH 8.0 |
| 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 16-18h at room temperature 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 column was washed with 2xPBX, 3M urea, 6mM imidazole, 5mM beta-mercaptoethanol pH 8.0 until A280 reached baseline. The protein was then eluted with 2xPBS, 2M urea, 5mM beta-mercaptoethanol, 400mM imidazole, pH 7.5 |
| Refolding Assay | Immunoassay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 40mg/L |
| Purity | |
| Notes | - similar protocol for enterotoxin A (TcdA), record 1479 |