Refolding Record:
| Protein | |
|---|---|
| Protein Name | Immulectin-3 |
| Abbreviated Name | IML-3 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Manduca sexta (Tobacco hawkmoth) (Tobacco hornworm |
| UniProt Accession | Q5UAW8 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 310 |
| Molecular Weight | 33867.5 |
| Pi | 5.85 |
| Molecular Weight | 33867.5 |
| Disulphides | Unknown |
| Full Sequence |
MEVLRGVVVLITVSIVQGSNVFRADYEYHASAGGWFKFHKVPADWHDARLMCDFEGAVLASPINVDVTDVLQNIINKIEHLSTGVHTGVHNTISPVVFNSIEGVPLSALPVRTRDMFTEEYSSGPHCARLIPQEGLVAGSCSDALPYICYKNKTAELSMTECGTVDKGYQLSAKTGHCYKFHNYGLPWSLAYLRCIAEGGQLAVINSAVEANVLKELLARYPTGLIKGGYAGGAFLGFHDWNNNNVWRTVNGQTLEEAGYANWGVTQPDSSVQNCGQMFRSGQLDDIGCAKVPFICEKHPNNIMPVPNNV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Yu XQ, Tracy ME, Ling E, Scholz FR, Trenczek T. (2005) Insect Biochem Mol Biol., 35, 285-295 |
| Project Aim | Drug Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | XL1-Blue |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | pGEMR-T |
| Expression Protocol | Expression of IML-3 and production of antibodies: The cDNA fragment encoding amino acid residues 18 to 310 (mature IML-3) (Fig. 1) was generated by PCR using primers LEXF1 (5′-TAG CCA TGG GAA GCA ATG TGT TTC-3′) and LEXR2 (5′-ATT AAG CTT AAT TAA ACG TTG TTT G-3′). The cDNA fragment was cloned into plasmid vector pGEMR-T (Promega) and the insert encoding IML-3 was excised by complete digestion with Nco I and Hind III. The digested insert was then separated by electrophoresis, recovered, and cloned into the Nco I/Hind III sites of expression vector H6pQE-60 (Lee et al., 1994). Recombinant IML-3 was expressed in E. coli strain XL1-Blue (Stratagene) as described previously (Yu et al., 1999). |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea |
| Refolding Buffer | 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM reduced glutathione, 0.02 mM oxidized glutathione, 5% glycerol, and 0.005% Tween-20, then in 1 M urea in the same buffer, and finally in the buffer without urea, glutathione and Tween-20 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 12 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2-0.02 mM |
| Refolding Protocol | Recombinant IML-3 was expressed as an inclusion body and was purified under denaturing conditions in 8 M urea using nickel-nitrilotriacetic acid (Ni-NTA) resins (Qiagen) following the manufacturer\'s instructions. Purified IML-3 in 8 M urea was renatured by three dialysis steps, first in 3 M urea in a buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM reduced glutathione, 0.02 mM oxidized glutathione, 5% glycerol, and 0.005% Tween-20, then in 1 M urea in the same buffer, and finally in the buffer without urea, glutathione and Tween-20. Each dialysis step was performed for at least 12 h at 4 °C. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol,tween 20 |
| Additives Concentration | 5%-0.005% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |