Refolding Record:
| Protein | |
|---|---|
| Protein Name | CtcC |
| Abbreviated Name | n/a |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Chlamydia trachomatis |
| UniProt Accession | O84474 |
| 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 | 386 |
| Molecular Weight | 43169.7 |
| Pi | 6.23 |
| Molecular Weight | 43169.7 |
| Disulphides | Unknown |
| Full Sequence |
MSIEHILIIDDDPHILALLSEILGARNFSVSSAPGVKQAIKQISNCPFDLIISDMNMPDGSGLDIIQYTKQHRPQTPILVITAFGTIQNAVEAMRFGAFNYLTKPFSPDALFTLIAKAEELQALQQDNLFLQSQGSSISHPLIAESPSMKQLLDKARRAANSSANIFVHGESGCGKENLSFFIHKHSPRSTKPYIKVNCAAIPDTLLESEFFGHEKGAFTGATTKKVGRFELAHQGTLLLDEITEIPIHLQAKLLRAIQEQEFEHIGGIKTLPVNIRFLATSNRDLEEAIETKVLRQDLYYRLSVISLHIPPLRDRKEDILPLAHYYLEKFCKMNNKPPKTLSLEAQRNLLDYSWPGNVRELSNVLERTVILENDPAITPSMLALL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Koo IC, Stephens RS. (2003) Biologycal Chemistry, 278, 17314-17319 |
| Project Aim | Undefined |
| Fusion | None |
| 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 | 2 h |
| Expression Vector | pET21b |
| Expression Protocol | The pET21b constructs were transformed into E. coli strain BL21(DE3). Bacterial cultures were grown at 37 °C in 100 ml of Luria broth with 100 µg/ml ampicillin. At OD600 of 0.6, cultures were induced with 1 mM isopropyl-1-thio--D-galactopyranoside for 2 h. Overexpressed recombinant CtcB and CtcC were found in insoluble inclusion bodies and purified under denaturing conditions. Induced E. coli were pelleted by centrifugation at 8000 × g for 10 min and resuspended in 50 ml of wash buffer (10 mM HEPES, pH 7.2, 5 mM EDTA, and 0.1% Triton X-100), sonicated briefly on ice, and centrifuged. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ni-NTA column |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 10 mM HEPES, pH 7.2, 5 mM EDTA, and 0.1% Triton X-100 |
| Solubilization Buffer | 5 ml of phosphate buffer, pH 6.8, with 6 M guanidine hydrochloride |
| Refolding Buffer | 30 mM Tris-HCl, pH 7.6, 200 mM KCl, 1 mM EDTA, 5 mM dithiothreitol, 10% (v/v) glycerol) containing 6 M urea |
| Pre-Refolding Purification | Ni-NTA column |
| Tag Cleaved | no tag |
| Refolding pH | 7.6 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 4 h |
| Redox Agent | DTT |
| Redox Agent Concentration | 5 mM |
| Refolding Protocol | Induced E. coli were pelleted by centrifugation at 8000 × g for 10 min and resuspended in 50 ml of wash buffer (10 mM HEPES, pH 7.2, 5 mM EDTA, and 0.1% Triton X-100), sonicated briefly on ice, and centrifuged. The pellet was washed three times with wash buffer and dissolved in 5 ml of phosphate buffer, pH 6.8, with 6 M guanidine hydrochloride for 1 h on ice. After a final centrifugation of 39,000 × g for 20 min, supernatant containing denatured recombinant protein was loaded on a nickel resin column according to manufacturer\'s instructions (Novagen, Inc.). Denatured protein was refolded by dialysis at 4 °C against refolding buffer (30 mM Tris-HCl, pH 7.6, 200 mM KCl, 1 mM EDTA, 5 mM dithiothreitol, 10% (v/v) glycerol) containing 6 M urea (13). The buffer was exchanged for decreasing concentrations of urea (3, 2, 0.5, 0, 0 M) for at least 4 h per buffer exchange. Refolded protein was divided into 50-µl aliquots and stored at 20 °C. |
| Refolding Assay | Phosphorylation,Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 10% v/v |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |