Refolding Record:
| Protein | |
|---|---|
| Protein Name | Aspartyl protease |
| Abbreviated Name | OsAsp1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Oryza sativa L. |
| UniProt Accession | A2ZC67 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 370 |
| Molecular Weight | 40033.1 |
| Pi | 9.05682 |
| Molecular Weight | 40033.1 |
| Disulphides | Unknown |
| Full Sequence |
PAKPYFLDIDTGST
LTWLQCDYPCINCNKVPHGLYKPELKYAVKCTEQRCADLYADLRKPMKCGPKNQCHYGIQ
YVGGSSIGVLIVDSFSLPASNGTNPTSIAFGCGYNQGKNNHNVPTPVNGILGLGRGKVTL
LSQLKSQGVITKHVLGHCISSKGKGFLFFGDAKVPTSGVTWSPMNREHKHYSPRQGTLQF
NSNSKPISAAPMEVIFDSGATYTYFALQPYHATLSVVKSTLSKECKFLTEVKEKDRALTV
CWKGKDKIRTIDEVKKCFRSLSLKFADGDKKATLEIPPEHYLIISQEGHVCLGILDGSKE
HPSLAGTNLIGGITMLDQMVIYDSERSLLGWVNYQCDRIPRSASAITSRL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bi Xuezhi , Khush GS, and Bennett J (2005) Acta Biochemica et Biophysica Sinica, 46, 87-98 |
| Project Aim | Folding |
| Fusion | N-terminal thioredoxin + 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 | 4hr |
| Expression Vector | pET32 EK/LIC |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Freeze/Thaw+Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 60 mM imidazole, 500mM NaCl, 20mM Tris-Cl, pH7.9, 6M Urea |
| Solubilization Buffer | 5 mM imidazole, 500 mM NaCl, 20 mM Tris-Cl, pH 7.9, 6 M urea |
| Refolding Buffer | 50 mM acetate buffer |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 3.5 |
| Refolding Temperature | 22.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | The OsAsp1 coding region excluding the sequence coding for the predicted N-terminal 23 amino residue signal peptide (http://www.cbs.dtu.dk/services/SignalP) was amplified by high fidelity Taq polymerase (Promega) PCR, and subcloned into pET32 EK/LIC expression vector (Novagen), in which proOsAsp1 was fused to the C-terminus of thioredoxin and a 6His tag according to the manufacturer’s manual. E. coli strain BL21 cells harboring the expression construct were cultured to an OD600 of 0.6, and protein expression was induced by 1 mM isopropyl 1-thio-?D-galactopyranoside (IPTG) added to the media, with further incubation at 30°C for 5 h. The thioredoxin–proOsAsp1 fusion protein was extracted in 1x binding buffer (containing 5 mM imidazole, 500 mM NaCl, 20 mM Tris–HCl, pH 7.9, 6 M urea), and affinity purified under denaturing conditions using the His·Bind purification kit (Novagen) according to the manufacturer’s instructions. Purified protein was refolded in 50 mM acetate buffer with pH 3.5, concentrated with PEG6000, dialyzed against the same buffer at 4°C overnight, and autolysed in 100 mM acetate buffer pH 3.5 at room temperature for 24 h. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | mixture |
| Notes | |