Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lectin |
| Abbreviated Name | ECL |
| SCOP Family | Legume lectins |
| Structure Notes | |
| Organism | Erythrina cristagalli |
| UniProt Accession | P83410 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Heterodimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 242 |
| Molecular Weight | 26231.3 |
| Pi | 4.72871 |
| Molecular Weight | 26231.3 |
| Disulphides | 0 |
| Full Sequence |
VETISFSFSEFEPGNDNLTLQGAALITQSGVLQLTKINQNGMPAWDSTGRTLYTKPVHMW
DSTTGTVASFETRFSFSIEQPYTRPLPADGLVFFMGPTKSKPAQGYGYLGVFNNSKQDNS
YQTLAVEFDTFSNPWDPPQVPHIGIDVNSIRSIKTQPFQLDNGQVANVVIKYDAPSKILH
VVLVYPSSGAIYTIAEIVDVKQVLPDWVDVGLSGATGAQRDAAETHDVYSWSFQASLPE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Stancombe PR, Alexander FC, Ling R, Matheson MA, Shone CC, Chaddock JA. (2003) Protein Expression and Purification, 30, 283-292 |
| Project Aim | Undefined |
| Fusion | None |
| 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 | 10-14h |
| Expression Vector | pMTL1015 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Freeze/Thaw-High Pressure Homogenization |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 100mM Tris-HCl, 1M NaCl, 1% Triton X-114, pH 8.0 |
| Solubilization Buffer | 10mM CAPS, 6M urea, pH 10.5 |
| Refolding Buffer | 10mM CAPS |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 10.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 66h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Escherichia coli BL21 cell paste was suspended in 150 ml suspension buffer (50 mM Tris, 0.15 M NaCl, and pH 8.0 + 0.3 mg/ml lysozyme) per litre culture, prior to freeze-thaw and homogenisation (Gaulin, 500 bar) in the presence of DNase (7 g/ml, 5 mM MnCl2). Following clarification by centrifugation (8000g, 30 min, 4 °C), the pellet was washed twice with 1% Triton X-114 in 100 mM Tris, 1 M NaCl, pH 8.0. The resultant inclusion body pellet was solubilised using solubilisation buffer (6 M urea, 10 mM CAPS, pH 10.5) at a final OD280 of 0.5?.725. After a minimum of three hours in solubilisation buffer, refolding was achieved by a step dilution between 1:10 and 1:18 in refolding buffer (10 mM CAPS, pH 10.5). After 30 min in refolding buffer, the pH was adjusted to pH 7.4 by addition of 0.01 volume 1 M Tris, pH 8.0, and HCl. The diluted material was left at 4 °C without mixing for a minimum of 66 h; prior to concentration by tangential flow (10 kDa membranes) to 4 L. The concentrated material was dialysed into column equilibration buffer (20 mM Tris, 0.15 M NaCl, 4 °C) supplemented with 1 mM MnCl2 + 1 mM CaCl2 and subsequently clarified by centrifugation (8000g, 30 min, 4 °C). The resultant supernatant was diluted 1:1 in column equilibration buffer, aliquoted into appropriately sized batches, and applied to a previously equilibrated immobilised lactose matrix (50 ml bed volume), and following extensive washing, recECL was eluted by the addition of 0.3 M lactose (in column equilibration buffer). |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 870mg/L |
| Purity | 90% |
| Notes | |