Refolding Record:
| Protein | |
|---|---|
| Protein Name | Urokinase-type plasminogen activator |
| Abbreviated Name | uPA |
| SCOP Family | Kringle Modules |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P00749 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 413 |
| Molecular Weight | 46385.8 |
| Pi | 8.69 |
| Molecular Weight | 46385.8 |
| Disulphides | >10 |
| Full Sequence |
SNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGKKPSSPPEELKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLMSPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLAL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hua ZC, Dong C, Zhu DX. (1996) Biochemical and Biophysical Research Com, 220, 131-136 |
| Project Aim | Over expression & Renaturation |
| 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 | pET3 |
| Expression Protocol | Protein expression. A culture from a single colony was grown at 37°C overnight and innoculated into LB medium containing 100􏰚g/ml ampicillin and 34g/ml chloramphenicol using 2% of the final growth volume, then grown at 37°C until the OD600 reached 0.6. The induction of pro-UK expression was begun by addition of 0.1mM IPTG. After expressed for 3 hours, bacteria were harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M urea, 50mM B-mercaptoethanol, 0.1M.Na phosphate buffer, pH7.5 |
| Refolding Buffer | 2.5M urea, 50mM Tris-HC1, 5mM EDTA, 10mM NaCl, 1.25mM GSH, 0.25mM GSSG, 10mM lysine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 18-24 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1.25/0.25 mM |
| Refolding Protocol | Denaturation and renaturation. Centrifuged cells from 60ml flask culture were suspended in 4ml 0.1M Na phosphate buffer, pH7.5 and disrupted by sonication. The suspension was centrifuged at 10,000rpm for 10 minutes and the collected pellet was solubilized in 8M urea, 50mM 􏰙-mercaptoethanol, 0.1M.Na phosphate buffer, pH7.5 at 4°C for 16 hours, then dialyzed against 8M urea, 1mM 􏰙-mercaptoethanol, 0.1M Na phosphate buffer, pH7.5 for 6 hours and renatured by 50-fold dilution in renaturation buffer at 4°C for 18–24 hours. After dialysis against 0.01M Na phosphate buffer, pH7.5, fibrinolytic activity was assayed on fibrin plate prepared as described by Ploug and Kieldgaard (18) |
| Refolding Assay | Protein activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |