Refolding Record:
| Protein | |
|---|---|
| Protein Name | Mannose-binding lectin |
| Abbreviated Name | n/a |
| SCOP Family | alpha-D-mannose-specific plant lectins |
| Structure Notes | |
| Organism | Dendrobium officinale |
| UniProt Accession | Q3L635 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 141 |
| Molecular Weight | 16005.0 |
| Pi | 6.32 |
| Molecular Weight | 16005.0 |
| Disulphides | Unknown |
| Full Sequence |
DNHLLPGDRLNPGNFLKQDRYMLIMQEDCNLVLYNLNKPEWATKTANRGSRCFVTLQSDGNFVIYDDHEERNEAIWASNTDGQNGNYVIILQKDGNLVLYSKPIFATGTNRFGSTAVVVAKRNRKAHFGVEQNIIEVTTNL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chen Z, Sun X, Tang K (2004) Toxicon, 45, 535-540 |
| Project Aim | Cloning and expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15 |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pQE30-doa2 |
| Expression Protocol | The resulting recombinant plasmid pQE30-doa2 was sequenced and transformed into E. coli M15 strain. M15 cell strains transformed with pQE30- doa2 were grown in LB (Luria-Bertani) medium containing 100 μg/ml ampicillin and 25 μg/ml kanamycin at 37 °C to an absorbance of 0.5–1.0 at 600 nm. Then the culture was induced by addition of 1 mM IPTG. The cells were harvested three hours after induction and centrifuged at 4 °C at 5000 rpm for 10 min |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5-1.0 = 600 |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | pH 8.0 buffer B containing 10 mM Tris–HCl, 0.1 M NaH2PO4, and 8 M urea |
| Solubilization Buffer | pH 6.3 buffer C containing 10 mM Tris–HCl, 0.1 M NaH2PO4, and 8 M urea |
| Refolding Buffer | 150 mM NaCl, 50 mM MgCl2 and 50 mM CaCl2 pH 5.4 |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 5.4 |
| Refolding Temperature | 28.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Some of the pellets were used to determinate the recombinant DOA2 protein solubility. Subsequently, other pellets were resolved in pH 8.0 buffer B containing 10 mM Tris–HCl, 0.1 M NaH2PO4, and 8 M urea. Following centrifugation at 4 °C at 15,000 rpm for 30 min, the supernatant was loaded into an equilibrated His-bond Ni Affinity Resin column (Watson, China), washed twice with buffer C (pH 6.3) and eluted twice with buffer E (pH 4.5). The eluates were collected respectively and all samples including the unbound fractions were analyzed by SDS-PAGE.DOA2 renatured by dialysis against dialysis buffer (150 mM NaCl, 50 mM MgCl2 and 50 mM CaCl2 pH 5.4). After incubation in the incubator at 28 °C for 24 h |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |