Refolding Record:
| Protein | |
|---|---|
| Protein Name | Pro-Falcipain-2 |
| Abbreviated Name | pro-Falcipain-2 |
| SCOP Family | Papain-like Cysteine Proteinases |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q9NBD4 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 287 |
| Molecular Weight | 32713.0 |
| Pi | 6.01482 |
| Molecular Weight | 32713.0 |
| Disulphides | Unknown |
| Full Sequence |
MRGSHHHHHH GKELNRFADLTYH EFKNKYLSLR SSKPLKNSKY LLDQMNYEEV IKKYRGEENF DHAAYDWRLH SGVTPVKDQK NCGSCWAFSS IGSVESQYAI RKNKLITLSE QELVDCSFKN YGCNGGLINN AFEDMIELGG ICPDGDYPYV SDAPNLCNID RCTEKYGIKN YLSVPDNKLK EALRFLGPIS ISVAVSDDFA FYKEGIFDGE CGDELNHAVM LVGFGMKEIV NPLTKKGEKH YYYIIKNSWG QQWGERGFIN IETDESGLMR KCGLGTDAFIPLIE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Sijwali PS, Brinen LS, Rosenthal PJ. (2001) Protein Expression and Purification, 22, 128-134 |
| 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 | M15(pREP4) |
| Expression Temp | 37.0 |
| Expression Time | 5h |
| Expression Vector | pQE30 |
| Expression Protocol | Cells were grown at 37degC to mid-log phase, at which point 0.25mM IPTG was added. Cells were grown a further 5h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 1:2.0 M urea, 20 mM TrisCl, 2.5% Triton X-100, pH 8.0 2: 20% sucrose, 20mM TrisCl, pH 8.0 |
| Solubilization Buffer | 6M guanidineHCl, 20 mM TrisCl, 500 mM NaCl, 10 mM imidazole, pH 8.0 |
| Refolding Buffer | 100 mM TrisCl, 1 mM EDTA, 30% glycerol, 250 mM L-arginine, 1 mM each of GSH and GSSG |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 9.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.12mg/ml |
| Refolding Time | 20h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1mM/1mM |
| Refolding Protocol | NiNTA-purified falcipain-2 was reduced with 10mM dithiothreitol (DTT) at 37C for 45 min. Refolding was carried out by diluting the protein 100-fold in 100 mM TrisCl, 1 mM EDTA, 30% glycerol, 250 mM L-arginine, 1 mM each of GSH and GSSG. The solution was stirred constantly for 20h at 4C. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine,Glycerol |
| Additives Concentration | 30%/250mM |
| Refolding Yield | |
| Purity | 91% |
| Notes | |