Refolding Record:
| Protein | |
|---|---|
| Protein Name | LMW Urokinase Plasminogen Activator |
| Abbreviated Name | LMW uPA |
| SCOP Family | Eukaryotic Proteases |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P00749 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Serine protease domain, aa.136-411 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 276 |
| Molecular Weight | 30962.5 |
| Pi | 8.47523 |
| Molecular Weight | 30962.5 |
| Disulphides | 6 |
| Full Sequence |
QVGLK PLVQECMVHD CADGKKPSSP PEELKFQCGQ KTLRPRFKII GGEFTTIENQ PWFAAIYRRH RGGSVTYVCG GSLMSPCWVI SATHCFIDYP KKEDYIVYLG RSRLNSNTQG
EMKFEVENLI LHKDYSADTL AHHNDIALLK IRSKEGRCAQ PSRTIQTICL PSMYNDPQFG TSCEITGFGK ENSTDYLYPE QLKMTVVKLI SHRECQQPHY YGSEVTTKML CAADPQWKTD SCQGDSGGPL VCSLQGRMTL TGIVSWGRGC ALKDKPGVYT R
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Fahey EM, Chaudhuri JB, Binding P. (2000) J Chromatography B, 737, 225-235 |
| Project Aim | Folding |
| Fusion | None |
| 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 | not stated |
| Expression Vector | not stated |
| Expression Protocol | no details provided |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | 1% TritonX-100, 25% sucrose, 50mM TrisHCl, pH 7.5, 5mM EDTA/0.5M urea, 50mM TrisHCl pH 7.5, 5mM EDTA |
| Solubilization Buffer | 6M GuHCl, 5mM DTT, 50mM TrisHCl pH 7.5, 5mM EDTA |
| Refolding Buffer | 3M urea, 50mM TrisHCl, 5mM EDTA, 0.5mM GSH, 0.5mM GSSG, pH 8.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 0.5mM/0.5mM |
| Refolding Protocol | 50g of E.coli cell paste containing over-expressed protein was suspended in 1L of lysis buffer (50mM TrisHCl pH 7.5, 5mM EDTA) and lysed by 6 passes through a high-pressure homogeniser at 41MPa. The inclusion bodies were collected by centrifugation, then were washed at room temperature (22degC) either for 2h in wash buffer, then further washes were also performed (1hr) to increase purity of the protein with 0.5M urea and 4M urea in 50mM Tris-HCl, pH 7.5. The final inclusion bodies pellet was solubilized in by homogenisation in solubilization buffer for 4min, followed by gentle agitation (22degC, 10h). The insoluble fraction was then removed by centrifugation (60min x 10400g) followed by filtration (0.22 micron). Size exclusion refolding was performed using a XK26/100 Sephacryl column cooled to 4degC. A 2ml volume of 8mg/ml denatured protein was injected through a static loop and eluted at a flow-rate of 0.5mg/min. The refolded protein was then acidified to pH 6.5 using 1M MES and loaded onto a 5ml Hi-Trap SP column at 5ml/min at room temperature. The column was washed with 2 column volumes of 50mM TrisHCl pH 7.5, 5mM EDTA, then eluted using a linear gradient of the same buffer with 1M NaCl. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |