Refolding Record:
| Protein | |
|---|---|
| Protein Name | Neurotrophin-3 |
| Abbreviated Name | NT-3 |
| SCOP Family | Neurotrophin |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P20783 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 112 |
| Molecular Weight | 12826.4 |
| Pi | 8.78 |
| Molecular Weight | 12826.4 |
| Disulphides | 3 |
| Full Sequence |
YAEHKSHRGEYS VCDSESLWVT DKSSAIDIRG HQVTVLGEIK TGNSPVKQYF YETRCKEARP VKNGCRGIDD KHWNSQCKTS QTYVRALTSE NNKLVGWRWI RIDTSCVCAL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Suenaga M, Ohmae H, Tsuji S, Itoh T, Nishimura O. (1998) Biotechnol. Appl. Biochem., 28, 119-124 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | MM294 |
| Expression Temp | 30.0 |
| Expression Time | 7h |
| Expression Vector | pET3c |
| Expression Protocol | Plasmid pENGFT103 was introduced into E. coli MM294(DE3) with plasmid pLysS and the cells were grown in 1 litre of LB medium supplemented with 50 mg/l ampicillin and 15 mg/l chloramphenicol at 30degC for 8 h. The culture was inoculated into 20 litres of M9 medium supplemented with 5% glucose, 1.5% Casamino acids (Difco, Detroit, MI, U.S.A.), 5 mg}l thiamin}HCl and iron(II) sulphate in a 50-litre fermentor. When the Klett value of the culture reached 500, isopropyl b-thiogalactoside was added to the culture to a final concentration of 100 mg/l, and fermentation was continued for an additional 7 h. The cells were collected by centrifugation and stored at -80deg C until use. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 10mM EDTA pH7.0 |
| Solubilization Buffer | 20mM citrate, 8M urea, pH 3.0 |
| Refolding Buffer | 0.1M phosphate, 0.2M L-arginine, 1mM GSH, 0.2mM GSSG |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 4 weeks |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1mM/0.2mM |
| Refolding Protocol | Refolding was initiated by a 20-fold dilution into refolding buffer. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.2mM |
| Refolding Yield | 10.7mg/L cultur |
| Purity | |
| Notes | |