Refolding Record:
| Protein | |
|---|---|
| Protein Name | Plasminogen Activator Inhibitor-1 |
| Abbreviated Name | PAI-1 |
| SCOP Family | Serpins |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P05121 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Multi-domain proteins (alpha and beta) |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 418 |
| Molecular Weight | 47186.9 |
| Pi | 6.67844 |
| Molecular Weight | 47186.9 |
| Disulphides | 0 |
| Full Sequence |
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSR VHHPPSY VAHLASDFGV RVFQQVAQAS KDRNVVFSPY GVASVLAMLQ LTTGGETQQQ IQAAMGFKID DKGMAPALRH LYKELMGPWN KDEISTTDAI FVQRDLKLVQ GFMPHFFRLF RSTVKQVDFS EVERARFIIN DWVKTHTKGM ISNLLGKGAV DQLTRLVLVN ALYFNGQWKT PFPDSSTHRR LFHKSDGSTV SVPMMAQTNK FNYTEFTTPD GHYYDILELP YHGDTLSMFI AAPYEKEVPL SALTNILSAQ LISHWKGNMT RLPRLLVLPK FSLETEVDLR KPLENLGMTD MFRQFQADFT SLSDQEPLHV AQALQKVKIE VNESGTVASS STAVIVSARM APEEIIMDRP FLFVVRHNPT GTVLFMGQVM EP FEA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hak-Joo Lee, Hana Im (2003) Protein Expression and Purification, 31, 99-107 |
| Project Aim | Undefined |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pRSET B |
| Expression Protocol | Cells were grown at 37degC in 1 L M9ZB medium (10 g Bacto-tryptone, 5 g NaCl, 1 g NH4Cl, 3 g KH2PO4, 6 g Na2HPO4, 4 g glucose, and 0.12 g MgSO4 per liter). When OD600 reached 0.6, expression was induced for 3 h at 37degC after addition of 0.1 mM IPTG. Cells were collected by centrifugation at 4500g for 10min and suspended in 40 ml of a buffer (20 mM sodium acetate, pH 5.6, 0.5 M NaCl, and 0.05% Tween 80). |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 4M guanidinium chloride, 20 mM sodium acetate, 1NaCl, 0.01% Tween 80, 0.05% beta-mercaptoethanol pH 5.6 |
| Refolding Buffer | 20mM sodium acetate, 1M NaCl, 0.01% Tween 80, pH 5.6 |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 5.6 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 1h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were disrupted using the sonicator, inclusion bodies were then obtained by high-speed centrifugation at 9800g for 30 min at 4degC. Inclusion bodies were dissolved in 40 ml of solubilization buffer. The protein was then refolded by dilution 25-fold into refolding buffer with slow stirring for 1 h at room temperature. The protein was then purified by Ni-NTA column chromatography. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |