Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lectin |
| Abbreviated Name | ECL |
| SCOP Family | Legume lectins |
| Structure Notes | |
| Organism | Common snowdrop (Galanthus nivalis) |
| UniProt Accession | P30617 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 157 |
| Molecular Weight | 16917.4 |
| Pi | 6.53532 |
| Molecular Weight | 16917.4 |
| Disulphides | 1 |
| Full Sequence |
MAKASLLILA AIFLGVITPS CLSDNILYSG ETLSTGEFLN YGSFVFIMQE DCNLVLYDVD KPIWATNTGG LSRSCFLSMQ TDGNLVVYNP SNKPIWASNT GGQNGNYVCI LQKDRNVVIY GTDRWATGTH TGLVGIPASP PSEKYPTAGK IKLVTAK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Longstaff M, Powell KS, Gatehouse JA, Raemaekers R, Newell CA, Hamilton WDO (1998) Eur J Biochem., 252, 59-65 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pET21c |
| Expression Protocol | 100ml L-broth with 100microg/ml ampicillin was inoculated with 0.5ml overnight culture. Cells were grown at 37degC until A600 reached 0.6, when expression was induced with 0.1mM IPTG and cells were grown for a further 3h. Cells were centrifuged and the cell pellet was stored at -20degC overnight. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Chemical |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | 20mM TrisHCl pH 7.5, 2mM EDTA |
| Solubilization Buffer | 5mM imidazole, 0.5M NaCl, 20mM TrisHCl pH 7.9, 6M urea |
| Refolding Buffer | 60mM imidazole, 0.5M NaCl, 20mM TrisHCl pH 7.9 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.9 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Inclusion bodies were prepared by cell lysis in 50mM TrisHCl pH 8.0, 25%(m/v) sucrose, 1mM EDTA, 10mg/ml lysozyme. 10mg/ml DNase, 10mM MgCl2 and 1mM MnCl2 were added. The inclusion bodies were precipitated by centrifugation (1300g, 10min) after addition of 2 volumes of 0.2M NaCl, 1% deoxycholate, 1% Nonidet P-40, 20mM TrisHCl pH 7.5, 2mM EDTA. The inclusion bodies werer then washed in wash buffer and dissolved in 4ml solubilization buffer. The protein was centrifuged (25000g, 20min) and loaded onto a 5ml Ni affinity column equilibrated with solubilization buffer. The column was washed with 10 volumes of solubilizatio buffer, then rinsed with refolding buffer containing decreasing amounts of urea - 2 volumes of refolding buffer each were used at 6, 4, 2, 1, 0.5, 0.25, and 0.125M urea. The refolded protein was then eluted with 4 volumes of refolding buffer with 300mM imidazole. Selected fractions were pooled, diluted to 100microg/ml and diaylised against 50mM TrisHCl pH 7.9, then concentrated to approximately 1ml. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |