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 | 412 |
| Molecular Weight | 46385.8 |
| Pi | 8.69 |
| Molecular Weight | 46385.8 |
| Disulphides | >10 |
| Full Sequence |
SNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGKKPSSPPEELKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLMSPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLAL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Zhu H, Liu W, Shi W, Xue Y, Kuai L, Ma Z. (2001) Chin Med J (Engl), 114, 186-190 |
| 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 | None |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | n/a |
| Expression Protocol | Bacterial cells for the production of recombinant pro-UK were cultured as previ ously reported in Ma Z, Hua ZC, Dong C, et al.High expression of human pro-urokinase gene in Es cherichia coli.Acta Biochem Biophys Sinica 1995;27:17-22. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50 mmol/L Tris-HCl, 5 mol/L urea, 5‰ Triton X-100, pH 7.5 |
| Solubilization Buffer | 6 mol/L Gudn-HCl, 50 mmol/L Tris-HCl, 10 mmol/L DTT, pH 7.5 |
| Refolding Buffer | 2.5 mol/L urea, 0.1 mol/L Tris-HCl, 1 mmol/L GSH, 0.05 g/L PEG 8000, 5% glycerol, pH 8.6-8.8 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.6 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | GSH |
| Redox Agent Concentration | 1 mM |
| Refolding Protocol | After centrifugation at 12 000 r/min for 20 min, th e pellets containing the inclusion bodies of pro-UK were washed once in washing b uffer (50 mmol/L Tris-HCl, 5 mol/L urea, 5‰ Triton X-100, pH 7.5) and drie d by acetone extraction.Ten mg inclusion body was dissolved in 1 ml of highly chaotropic and reduced solution (6 mol/L Gudn-HCl, 50 mmol/L Tris-HCl, 10 mmol/L DTT, pH 7.5) at 37℃ for 2 h.Any insoluble materials were removed by centrifugation.Renaturation Determination of basic renaturation conditions Denatured pro-UK (0.9 mg/ml) described above was diluted 100-fold by drippin g it slowly into stirring renaturation solution, and kept at 4℃ for 24 h to al low renaturation.The effects of various pH, temperature, denaturant concentrat ion, protein concentration, ratio of reduced and oxidized thiol reagents, and di fferent kinds of additives were compared to determine the basic pro-UK renatura tion conditions.Step-wise renaturation Denatured pro-UK (0.9 mg/ml) was divided into three parts, and was diluted 10 0-fold by dripping it slowly, at 60 min intervals, into stirring renaturation solution (2.5 mol/L urea, 0.1 mol/L Tris-HCl, 1 mmol/L GSH, 0.05 g/L PEG 8000, 5% glycerol, pH 8.6-8.8).It was then kept at 4℃ for 24 h to allow re naturation. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 5% |
| Refolding Yield | 20%-30% |
| Purity | n/a |
| Notes | n/a |