Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lectin |
| Abbreviated Name | ECL |
| SCOP Family | Legume lectins |
| Structure Notes | |
| Organism | Dolichos biflorus seeds |
| UniProt Accession | P05045 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Tetramer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 253 |
| Molecular Weight | 27129.2 |
| Pi | 4.83 |
| Molecular Weight | 27129.2 |
| Disulphides | Unknown |
| Full Sequence |
ANIQSFSFKNFNSPSFILQGDATVSSGKLQLTKVKENGIPTPSSLGRAFYSSPIQIYDKSTGAVASWATSFTVKISAPSKASFADGIAFALVPVGSEPRRNGGYLGVFDSDVYNNSAQTVAVEFDTFSNSGWDPSMKHIGIDVNSIKSIATVSWDLANGENAEILITYNAATSLLVASLVHPSRRTSYILSERVDITNELPEYVSVGFSATTGLSEGYIETHDVLSWSFASKLPDDSTAEPLDLASYLVRNVL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Cheng DH, Shen Q, Qian J, Qian Z, Ye QZ. (1994) Archives Biochemistry and Biophysics, 313, 346-350 |
| Project Aim | Identification and Characterization |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 1 h |
| Expression Vector | pET-8c |
| Expression Protocol | Expression of lectin. Transformed E coli were grown overnight at 37 c in LB medium containing 100 ug/ml ampicillin. the cultures were diluted 1;10 with fresh medium and grown to an OD600 of 0.6-1.0 by shaking for 1 h at 37 c after which IPTG was added to a final concentration of 0.4 mM. At various time points the OD600 was read and aliquots were removed and mixed with an equal volume of sample buffer for gel electrophoresis. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6-1.0 = 600 |
| Cell Disruption Method | None |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | Tris HCl 50 mM pH 7.3, |
| Solubilization Buffer | Tris HCl 50 mM pH 7.3,buffer containing urea 8 mol/L |
| Refolding Buffer | Tris HCl 50 mM pH 7.3 and than against PBS |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.3 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | The isolated inclusion bodies from the above cells were washed with 40 mM Tris-HCl,pH7.3 , and resuspended to 1/50 of the original volume of the cells with 50 mM Tris-HCl,pH7.3 . This rapid dilution step was found to be essential to prevent extensive precipitation. After dilution, the sample was dialyzed extensively against 50 mM Tris-HCl,pH7.3 and than against PBS. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | The temperature and time was not specified assumed room it was temperature. |