Refolding Record:
| Protein | |
|---|---|
| Protein Name | Tissue-type plasminogen activator |
| Abbreviated Name | t-PA |
| SCOP Family | Kringle Modules |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P00750 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 562 |
| Molecular Weight | 62917.1 |
| Pi | 8.13 |
| Molecular Weight | 62917.1 |
| Disulphides | 3 |
| Full Sequence |
MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Wang LF, Hum WT, Kalyan NK, Lee SG, Hung PP, Doi RH (1989) Gene, 84, 127-133 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Bacillus subtilis |
| Expression Strain | DB104 |
| Expression Temp | 37.0 |
| Expression Time | unknown |
| Expression Vector | pBL1 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | unknown |
| Solubilization Buffer | 50mM Tris-HCl, 6M guanidinium chloride, 20mM betamercaptoethanol, pH 8.0 |
| Refolding Buffer | 50mM Tris-HCl, 1M guanidinium chloride, 0.005% Tween-80, 1.25mM GSSG, 0.25mM GSH |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 9.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 32h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | The inclusion body pellet was washed once with water and solubilized in 50mM Tris-HCl, 6M guanidinium chloride, 20mM betamercaptoethanol, pH 8.0 at 4C for 24h with gentle shaking. Following clarification with centrifugation the supernatant was slowly diluted with 5 litres of 50mM Tris-HCl, 1M guanidinium chloride, 0.005% Tween-80, 1.25mM GSSG, 0.25mM GSH (pH 9.0). The mixture was allowed to stand at 4C for 16h and then further diluted with 2 vols of water. After standing for another 16h at 4C, the protein was purified. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | <10% |
| Purity | |
| Notes | |