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 | R275D |
| Chain Length | 354 |
| Molecular Weight | 39386.4 |
| Pi | 6.34402 |
| Molecular Weight | 39386.4 |
| Disulphides | 3 |
| Full Sequence |
SE GNSDCYFGNG SAYRGTHSLT ESGASCLPWN SMILIGKVYT AQNPSAQALG LGKHNYCRNP DGDAKPWCHV LKNRRLTWEY CDVPSCSTCG LRQYSQPQFD 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 | 35.0 |
| Expression Time | 3h |
| Expression Vector | p51H |
| Expression Protocol | Cells were grown in 100L of modified M9 broth containing 0.7% yeast extract, 0.155% Leu, 0.155% Ile, 0.155% Pro, 0.5% glucose, 50microg/ml vitamin B1 and 25microg/ml ampicillin. Cells were grown at 35degC with vigorous stirring . 0.5% glucose was added every 30min after 2.5h and 1% glucose was added after 7h. When A600 reached 15, protein expression was induced by the addition of 10microg/ml beta-indoleacrylic acid every 30min for 3 hours. The cells were harvested when A600 reached 30. |
| Method of Induction | beta-indoleacrylic acid |
| Cell Density at Induction | OD 600 = 15 |
| Cell Disruption Method | None |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 10mM TrisHCl pH 8.0, 0.5% Triton X-100, 10mM EDTA |
| Solubilization Buffer | 10mM NH4OAc pH 9.5, 8M urea |
| Refolding Buffer | 1: 10mM NH4OAc pH 9.5, 0.4mM cysteine, 0.04mM cystine, 2: 20mM TrisHCl pH 8.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 40h |
| Redox Agent | Cysteine/Cystine |
| Redox Agent Concentration | 0.4mM/0.04mM |
| Refolding Protocol | Pelleted cells corresponding to 2.0L of broth were suspended in 1L PBS, treated with 200mg lysozyme at 4degC for 2h, dispersed using a Biotron blender and centrifuged. The pellet was washed twice with wash buffer then dissolved in solubilization buffer. After centrifugation, the supernatant was dialyzed against 160L of refolding buffer 1 at 25degC for 16h. The protein was then purified further using a QAE-Toyopearl column equilibrated with 2M urea, 20mM TrisHCl pH 8.0. The protein was eluted with equilibration buffer containing 1M NaCl. Selected fractions were then dialyzed against 40L of Refolding buffer 2 for 16h at 4degC. The protein was then purified further using another QAE-Toyopearl column, this time eluting with a gradient of 0-1M NaCl in 20mM TrisHCl pH 8.0 and 2Murea. The protein was then dialysed against 20L of refolding buffer 2, and the protein was then purified further using a p-aminobenzamidine sepharose 4B column. |
| Refolding Assay | ELISA |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |