Refolding Record:
| Protein | |
|---|---|
| Protein Name | Plasminogen |
| Abbreviated Name | Pg |
| SCOP Family | Kringle Modules |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P00747 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Kringle 5 domain aa.481-560 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 80 |
| Molecular Weight | 9000.0 |
| Pi | 8.28 |
| Molecular Weight | 9000.0 |
| Disulphides | 3 |
| Full Sequence |
CMFGNGKGYR GKRATTVTGT PCQDWAAQEP HRHSIFTPET NPRAGLEKNY CRNPDGDVGG PWCYTTNPRK LYDYCDVPQC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lu H, Zhang H, Wang Q, Yuan H, He W, Zhao Z, Li Y. (2001) Current Microbiology, 42, 211-216 |
| Project Aim | Undefined |
| Fusion | N-terminal interferon gamma |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 42.0 |
| Expression Time | 3h |
| Expression Vector | pGEM-T |
| Expression Protocol | An overnight culture of E. coli strain BL21 transformants, containing the recombinant plasmid, was diluted 50-fold into 50 ml of LB medium containing ampicillin, and incubation at 37degC was continued for 10 h with shaking at 220 rpm and sampling at 1-h intervals. Optical density was measured at 600 nm. Cultures at different densities were induced at 42degC for another 3 h. |
| Method of Induction | Temperature Shift |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris-HCl, 100mM NaCl, 1mM EDTA, 0.5% Triton X100, pH 8.0 |
| Solubilization Buffer | 8M urea, 2mM EDTA, 0.4% betamercaptoethanol |
| Refolding Buffer | 50mM Tris-HCl, 1M urea, 0.5M L-Arginine, 2% glycine, 10mM GSH, 2mM GSSG |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no |
| Refolding pH | 9.0 |
| Refolding Temperature | 26.0 |
| Protein Concentration | |
| Refolding Time | 16-20h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 10mM/2mM |
| Refolding Protocol | Refolding was performed under aeration, with shaking at 150rpm at 26C for 16-20h. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine,Glycine |
| Additives Concentration | 2%/0.5M |
| Refolding Yield | 71% |
| Purity | |
| Notes | |