Refolding Record:
| Protein | |
|---|---|
| Protein Name | Thrombopoietin |
| Abbreviated Name | TPO |
| SCOP Family | Short Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P40225 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 154 |
| Molecular Weight | 16614.6 |
| Pi | 9.43 |
| Molecular Weight | 16614.6 |
| Disulphides | Unknown |
| Full Sequence |
SPAPPACDLRVLSKLLRDSHVLHSRLSQCPEVHPLPTPVLLPAVDFSLGEWKTQMEETKAQDILGAVTLLLEGVMAARGQLGPTCLSSLLGQLSGQVRLLLGALQSLLGTQLPPQGRTTAHKDPNAIFLSFQHLLRGKVRFLMLVGGSTLCVRR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hou J, Zhan H. (1998) Cytokine, 10, 319-330 |
| Project Aim | Identification and Characterization |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | n/a |
| Expression Protocol | E. coli clone 16 was grown in 10 ml TB containing 100 mg/ml ampicillin at 37°C with vigorous shaking. Fifteen hours later the culture was transferred to 1 litre prewarmed same medium and allowed to grow to OD600 of 0.8–1.0. IPTG was added to a final concentration of 1 mM and the culture was continued for another 3 h. The cells were harvested by centrifugation and washed once with TE. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | deionized wate |
| Solubilization Buffer | 2% sarkosyl, 50 mM CuSO4 and 20 mM Tris–HCl, pH 8.0 |
| Refolding Buffer | 6 M guanidine, 20 mM Na3 PO4, pH 6.5 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 6.5 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cells were resuspended in TE of 10 volumes of cell pellet, which was then incubated with 100 mg/ml lysozyme at room temperature for 1 h, followed by incubation with 5 mM MgCl2 and 1 mg/ml DNase I for 10 min at room temperature. The solution was cooled on ice and pulse-sonicated (checked under microscope for cell lysis). Inclusion bodies were pelleted by centrifugation at 1000 × g for 15 min and the supernatant was discarded. The pellet was washed with deionized water repeatedly until it turned clear greyish (inclusion body). The pellet was resuspended in 50 ml of 1% deoxycholate, 5 mM EDTA, 5 mM dithiothreitol (DTT) and 50 mM Tris–HCl, pH 9.0. Following stirring at room temperature for 20 min, the solution was centrifuged at 10 000 × g for 30 min and the resulted pellet was solubilized in 50 ml of 2% sarkosyl, 50 mM CuSO4 and 20 mM Tris–HCl, pH 8.0. The solution was stirred overnight at room temperature and then centrifuged at 20 000 × g for 30 min. The supernatant was cooled on ice and then 150 ml acetone was added, which was followed by stirring the solution for 30 min and centrifugation at 5000 × g for 30 min. The pellet was dissolved in 15 ml of 6 M guanidine, 20 mM Na3 PO4, pH 6.5 and was then loaded onto an 100 ml G-25 column. The sample was eluted with 20 mM Na3 PO4. Further purification was performed as follows: the sample from the G-25 column was loaded onto a SP-Sepharose HP column, after being washed thoroughly with 20 mM Na3 PO4, pH 6.5, the sample was eluted with a NaCl gradient from 0 to 1.0 M in 20 mM Na3 PO4, pH 6.5 over 20 column volumes. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |