Refolding Record:
| Protein | |
|---|---|
| Protein Name | C-terminal processing protease of the D1 protein |
| Abbreviated Name | CtpA |
| SCOP Family | Tail specific protease PDZ domain |
| Structure Notes | |
| Organism | Spinacia oleracea |
| UniProt Accession | Q36792 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 539 |
| Molecular Weight | 59334.8 |
| Pi | 8.84 |
| Molecular Weight | 59334.8 |
| Disulphides | Unknown |
| Full Sequence |
MEVISRLTFASVSYPFISSNLNPTPMLNSFNFRVLSWNSAPTNVAEAHLHRLLLRKLNPANDRVVGISNFGCSCRLDLWPSWRRHKRLFFQNGVSTIRWEVKKCSPKFYKIVSNYEKCKRHIYVPSVRLVVGVVLLMSVSVALNQDPSWSLSEENRIFLEAWRTIDRAYVDKTFNGQSWFRYRENALRNEPMNSREETYTAIRKMVATLNDPFTRFLEPEKLKSLRSGTQSSLTGVGISIGPTAVDQSSTGLVVISATPGAPASRAGILPGDVILAIDDASTDKMGIYEAANILQGPDGSSVDLTICSRDEIKHVVLKRERITLSPVKSRLCEMPGSAKDAPPKVGYIKLTTFTENASDAVKEAIETLRSNNVNAFVLDLRDNSGGLFPEGIEIAKIWLNKGVIVYICDSRGVRDIYDVEGSSAVAGSEPLVVLVNKGTASASEILAGALKDKKRAVVFGEPTYGKGKIQSVFELSDGSGLAVTVARYETPAHTDIGKVGIKPDHPLPASFPKDENDFCTCVQDPSSTCYLNGVQLFSR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Fabbri BJ, Duff SM, Remsen EE, Chen YC, Anderson JC, CaJacob CA. (2005) Pest Manag Sci., 61, 682-90 |
| Project Aim | Over expression & Renaturation |
| Fusion | N-terminal hexahistidine tag |
| 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 | 00 |
| Expression Vector | pET30a |
| Expression Protocol | Recombinant CtpA was expressed in E coli strain BL21(DE3) by induction with 5 mM IPTG at 37 ◦ C. CtpA was efficiently produced at levels of 5 – 10% of total SDS-soluble protein as estimated from visual inspection of a Coomassie Blue stained SDS-PAGE gel; however, almost all of the recombinant protein was found in inclusion bodies. Cells were collected by centrifugation, and disrupted by three passes through a microfluidizer. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Microfluidizer |
| Lytic Agent | None |
| Pre-Refolding Purification | Ni-NTA agrose chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | distilled water |
| Solubilization Buffer | guanidine-HCl 6 M; 100 mM Na-HEPES, pH 7.5 |
| Refolding Buffer | Na-HEPES, pH 7.7, 50 mM; urea 6 M; sodium chloride 500 mM; glycerol 200 ml/ liter |
| Pre-Refolding Purification | Ni-NTA agrose chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.7 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 2 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | To recover active CtpA, washed inclusion bodies were solubilized in buffer E (guanidine-HCl 6 M; Na- HEPES, pH 7.5, 100 mM). Insoluble material was removed by centrifugation for 15 min at 30 000g, followed by filtration through a 0.22-µm membrane. A nickel-affinity resin (Qiagen Ni-NTA Superflow) previously equilibrated in buffer E was added to bind the 6 × His tagged protein. Chromatography steps were carried out by gravity at room temperature. The resin was added to a chromatography column, and washed with additional buffer E until the absorbance at 280 nm was below 0.02. The resin was then washed with buffer F (Na-HEPES, pH 7.7, 50 mM; urea 6 M; sodium chloride 500 mM; glycerol 200 ml liter−1 ) until the absorbance at 280 nm was below 0.02. CtpA was refolded on the column by running a gradient of six column volumes from buffer F to buffer G (same as buffer F, but with no urea) over 2 h. CtpA was then eluted with a gradient of 0 – 500 mM imidazole in buffer G. The most active fractions eluted in the range of 100 – 250 mM imidazole; these were pooled and concentrated by precipitation by addition of ammonium sulfate to 65% saturation at 4 ◦ C. The collected precipitates were dissolved in a minimum amount of buffer H (Na-HEPES, pH 7.7, 50 mM; sodium chloride 100 mM; EDTA 1 mM; Tween-80 0.1 g liter−1 ; glycerol 100 ml liter−1 ). Insoluble material was removed by centrifugation at 30 000g for 30 min at 4 ◦ C, and the protein stored at −80 ◦ C. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 200 ml /liter |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |