Refolding Record:
| Protein | |
|---|---|
| Protein Name | Canstatin |
| Abbreviated Name | n/a |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | P08122 |
| 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 | 1525 |
| Molecular Weight | 149090.0 |
| Pi | 8.16 |
| Molecular Weight | 149090.0 |
| Disulphides | 6 |
| Full Sequence |
GEPGEPGLVGYQGPPGRPGPIGQMGPMGAPGRPGPPGPPGPKGQPGNRGLGFYGQKGEKGDIGQPGPNGIPSDITLVGPTTSTIHPDLYKGEKGDEGEQGIPGVISKGEEGIMGFPGIRGFPGLDGEKGVVGQKGSRGLDGFQGPSGPRGPKGERGEQGPPGPSVYSPHPSLAKGARGDPGFQGAHGEPGSRGEPGEPGTAGPPGPSVGDEDSMRGLPGEMGPKGFSGEPGSPARYLGPPGADGRPGPQGVPGPAGPPGPDGFLFGLKGSEGRVGYPGPSGFPGTRGQKGWKGEAGDCQCGQVIGGLPGLPGPKGFPGVNGELGKKGDQGDPGLHGIPGFPGFKGAPGVAGAPGPKGIKGDSRTITTKGERGQPGIPGVHGMKGDDGVPGRDGLDGFPGLPGPPGDGIKGPPGDAGLPGVPGTKGFPGDIGPPGQGLPGPKGERGFPGDAGLPGPPGFPGPPGPPGTPGQRDCDTGVKRPIGGGQQVVVQPGCIEGPTGSPGQPGPPGPTGAKGVRGMPGFPGASGEQGLKGFPGDPGREGFPGPPGFMGPRGSKGTTGLPGPDGPPGPIGLPGPAGPPGDRGIPGEVLGAQPGTRGDAGLPGQPGLKGLPGETGAPGFRGSQGMPGMPGLKGQPGFPGPSGQPGQSGPPGQHGFPGTPGREGPLGQPGSPGLGGLPGDRGEPGDPGVPGPVGMKGLSGDRGDAGMSGERGHPGSPGFKGMAGMPGIPGQKGDRGSPGMDGFQGMLGLKGRQGFPGTKGEAGFFGVPGLKGLPGEPGVKGNRGDRGPPGPPPLILPGMKDIKGEKGDEGPMGLKGYLGLKGIQGMPGVPGVSGFPGLPGRPGFIKGVKGDIGVPGTPGLPGFPGVSGPPGITGFPGFTGSRGEKGTPGVAGVFGETGPTGDFGDIGDTVDLPGSPGLKGERGITGIPGLKGFFGEKGAAGDIGFPGITGMAGAQGSPGLKGQTGFPGLTGLQGPQGEPGRIGIPGDKGDFGWPGVPGLPGFPGIRGISGLHGLPGTKGFPGSPGVDAHGDPGFPGPTGDRGDRGEANTLPGPVGVPGQKGERGTPGERGPAGSPGLQGFPGISPPSNISGSPGDVGAPGIFGLQGYQGPPGPPGPNALPGIKGDEGSSGAAGFPGQKGWVGDPGPQGQPGVLGLPGEKGPKGEQGFMGNTGPSGAVGDRGPKGPKGDQGFPGAPGSMGSPGIPGIPQKIAVQPGTLGPQGRRGLPGALGEIGPQGPPGDPGFRGAPGKAGPQGRGGVSAVPGFRGDQGPMGHQGPVGQEGEPGRPGSPGLPGMPGRSVSIGYLLVKHSQTDQEPMCPVGMNKLWSGYSLLYFEGQEKAHNQDLGLAGSCLARFSTMPFLYCNPGDVCYYASRNDKSYWLSTTAPLPMMPVAEEEIKPYISRCSVCEAPAVAIAVHSQDTSIPHCPAGWRSLWIGYSFLMHTAAGDEGGGQSLVSPGSCLEDFRATPFIECNGGRGTCHYFANKYSFWLTTIPEQNFQSTPSADTLKAGLIRTHISRCQVCMKNL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hou WH, Wang TY, Yuan BM, Chai YR, Jia YL, Tian F, Wang JM, Xue LX. (2004) Acta Biochemica et Biophysica Sinica, 36, 845-850 |
| Project Aim | Undefined |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15 |
| Expression Temp | 0.0 |
| Expression Time | 4 h |
| Expression Vector | pQE-mCan |
| Expression Protocol | The constructed expression vector pQE-mCan was transformed into E. coli M15 for expression. E. coli M15 was cultured in LB medium for approximately 2 h until the culture reached an A 600 of 0.6. Subsequently, recombinant mouse canstatin expression was induced by the addition of IPTG to a final concentration of 0.8 mM. After a 4 h induction, E. coli M15 cells were harvested by centrifugation at 5000 g, lysed by using the lysing buffer containing 8 M urea, 0.1 M NaH 2PO4, 0.01 M Tris-HCl, pH 8.0, and sonicated briefly. The mixture was centrifuged at 8000 g for 30 min. |
| 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 | 20 mM imidazole in 8 M urea, 0.1 M NaH2PO4, 0.01M Tris-HCl, pH 8.0 |
| Solubilization Buffer | 20 mM imidazole in 8 M urea, 0.1 M NaH2PO4, 0.01M Tris-HCl, pH 8.0 |
| Refolding Buffer | 20 mM Tris-HCl, pH 8.5 and 1 mM reduced glutathione, 0.2 mM oxidized glutathione in 20 mM Tris-HCl, pH 8.5 |
| Pre-Refolding Purification | Ni-NTA column |
| Tag Cleaved | yes |
| Refolding pH | 8.5 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1/0.2 mM |
| Refolding Protocol | The vector pMD18T-mCan containing the cDNA of mouse canstatin was constructed in our laboratory previThe mixture was centrifuged at 8000 g for 30 min. The supernatant fraction was applied onto the Ni-NTA spin column, and nonspecifically bound proteins were removed by washing with 20 mM imidazole in 8 M urea, 0.1 M NaH2PO4, 0.01M Tris-HCl, pH 8.0. The recombinant protein with His 6-tag was eluted by 250 mM imidazole in 0.1 M NaH 2PO4, 0.01 M Tris-HCl, pH 8.0, and refolded by dialysis against 20 mM Tris-HCl, pH 8.5 and 1 mM reduced glutathione, 0.2 mM oxidized glutathione in 20 mM Tris-HCl, pH 8.5. Protein concentration was determined by the bicinchoninic acid (BCA) assay. Refolded recombinant mouse canstatin was ready for use of the following experiments. All the above procedures were performed as described previously [8]. |
| Refolding Assay | Phosphorylation,Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | The temperature is not stated |