Refolding Record:
| Protein | |
|---|---|
| Protein Name | Falcipain-2(prime) |
| Abbreviated Name | Falcipain-2(prime) |
| SCOP Family | Papain-like Cysteine Proteinases |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q56CY9 |
| 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 | 307 |
| Molecular Weight | 34787.1 |
| Pi | 5.67762 |
| Molecular Weight | 34787.1 |
| Disulphides | Unknown |
| Full Sequence |
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSR FADLTYHEF KSKYLTLRSS KPLKNSKYLL DQINYDAVIK KYKGNENFDH AAYDWRLHSG VTPVKDQKNC GSCWAFSSIG SVESQYAIRK
NKLITLSEQE LVDCSFKNYG CNGGLINNAF EDMIELGGIC TDDDYPYVSD APNLCNIDRC TEKYGIKNYL SVPDNKLKEA LRFLGPISIS IAVSDDFPFY KEGIFDGECG DELNHAVMLV GFGMKEIVNP LTKKGEKHYY YIIKNSWGQQ WGERGFINIE TDESGLMRKC GLGTDAFIPL IE
|
| Notes | Mature protease domain, 271a.a. long |
| Expression | |
|---|---|
| Report | Singh N, Sijwali PS, Pandey KC, Rosenthal PJ (2006) Experimental Parasitology, 112, 187-192 |
| Project Aim | Functional Studies |
| 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(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 5h |
| Expression Vector | pRSET-B |
| Expression Protocol | Cells were incubated at 37degC until mid-log phase, at which time 0.25mM IPTG was added and growth was continued for 5h at 37degC. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| 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 TrisHCl, 2.5% Triton X-100, pH 8.0, 2: 20% sucrose, 20mM TrisHCl, pH 8.0 |
| Solubilization Buffer | 6M GdnHCl, 20 mM TrisHCl, 500 mM NaCl, 10 mM imidazole, pH 8.0 |
| Refolding Buffer | 100 mM TrisHCl, 1 mM EDTA, 30% glycerol, 250 mM L-arginine, 1 mM GSH, 1mM 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,1mM/1mM |
| Refolding Protocol | Harvested cells were washed with ice-cold 100mM TrisHCl, 10mM EDTA, pH 7.4 (5ml/g wet weight cells), sonicated and centrifuged (12000g, 30min, 4degC). The pellet was washed twice with wash buffer 1, then twice with wash buffer 2. The solution was then centrifuged (17000g, 30min, 4degC) and dissolved in solubilization buffer (5ml/g inclusion body pellet) for 60min at room temperature with gentle stirring. The solution was then centrifuged (27000g, 30min, 4degC) and the supernatant was was purified using Ni-NTA resin under denaturing conditions. The Ni-NTA-purified protein was reduced with 10mM dithiothreitol (DTT) at 37degC for 45 min, then refolding was carried out by diluting the protein 100-fold in refolding buffer. The solution was stirred constantly for 20h at 4degC. |
| Refolding Assay | Enzyme activity,Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine,Glycerol |
| Additives Concentration | 30%/250mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | Refolding protocol based on that of refolding record 1328 - Pro-falcipain 2, Sijwali et al., ( 2001) Protein Expression and Purification, 22, 128-134 |