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 | n |
| Domain | Kringle2 + protease domain aa.174-527 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 354 |
| Molecular Weight | 39427.5 |
| Pi | 6.65238 |
| Molecular Weight | 39427.5 |
| Disulphides | 3 |
| Full Sequence |
SE GNSDCYFGNG SAYRGTHSLT ESGASCLPWN SMILIGKVYT AQNPSAQALG LGKHNYCRNP DGDAKPWCHV LKNRRLTWEY CDVPSCSTCG LRQYSQPQFR IKGGLFADIA SHPWQAAIFA KHRRSPGERF LCGGILISSC WILSAAHCFQ ERFPPHHLTV ILGRTYRVVP GEEEQKFEVE KYIVHKEFDD DTYDNDIALL QLKSDSSRCA QESSVVRTVC LPPADLQLPD WTECELSGYG
KHEALSPFYS ERLKEAHVRL YPSSRCTSQH LLNRTVTDNM LCAGDTRSGG PQANLHDACQ GDSGGPLVCL NDGRMTLVGI ISWGLGCGQK DVPGVYTKVT NYLDWIRDNM RP
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Saito Y, Ishii Y, Sasaki H, Hayashi M, Fujimura T, Imai Y, Nakamura S, Suzuki S, Notani J, Asada T, Horiai H, Kobayashi M, Niwa M (1994) Biotechnol Prog, 10, 472 |
| Project Aim | Drug Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | HB101 |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | p51H |
| Expression Protocol | Cells were cultivated in 400ml M9Ca broth - see Saito et al. J.Biochem 1987, v101, p 123-134. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M urea |
| Refolding Buffer | 10mM NH4OAc, 2mM GSH, 0.2mM GSSG |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 9.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2mM/0.2mM,2mM/0.2mM |
| Refolding Protocol | Harvested inclusion bodies were dissolved in 120ml solubilization buffer and divided into seven portions. Each solution was dialyzed against 1.0 of buffer under different pH conditions (pH 6-12). Maximum refolding was achieved with pH 9.0-10.0. The buffer was centrifuged to remove insoluble material. |
| Refolding Assay | ELISA |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |