Refolding Record:
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 100 |
| Molecular Weight | 11266.9 |
| Pi | 5.63 |
| Molecular Weight | 11266.9 |
| Disulphides | 3 |
| Full Sequence |
LKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hillger F, Herr G, Rudolph R, Schwarz E. (2005) Biologycal Chemistry, 280, 14974-14980 |
| Project Aim | Biophysical Studies |
| Fusion | N-terminal hexahistidine tag |
| 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 | 3 h |
| Expression Vector | pET11a |
| Expression Protocol | For fermentation, the following medium was used: 54 g/liter yeast extract (OHLY KAV; DHW, Cologne, Germany), 0.54 g/liter NH4Cl, and 12 g/liter glycerol. The pH was adjusted with 1 M K2HPO4 to 7.0–7.4. Addition of 2.8 mM MgSO4, 0.01% thiamine, 0.1% ampicillin, and 0.05% kanamycin occurred by sterile filters. Feeding was started at A600 = 20. At A600 = 60, induction of gene expression was started by addition of 1 mM isopropyl-1-thio--D-galactopyranoside. After an additional 3 h of cultivation, cells were harvested by centrifugaion. Pellets were shock-frozen and stored at –80 °C. |
| Method of Induction | IPTG |
| 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 | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 5 M guanidinium chloride |
| Refolding Buffer | 0.1 M Tris/Cl, pH 8.0, 1 M L-arginine, 5 mM EDTA, 5 mM oxidized glutathione, and 2 mM reduced glutathione |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2/5 mM |
| Refolding Protocol | Protein Refolding and Purification—Inclusion body (IB) isolation and solubilization were performed according to Rudolph et al. (24). Renaturation of proBMP-2 and BMP-2 was carried out by 1:100 dilutions of IB protein solubilized in 5 M guanidinium chloride into refolding buffer (0.1 M Tris/Cl, pH 8.0, 1 M L-arginine, 5 mM EDTA, 5 mM oxidized glutathione, and 2 mM reduced glutathione). Prior to use, the buffer was degassed and pre-chilled to 10 °C. Protein concentration during renaturation was 3 µM. After renaturation (3–14 days), both proBMP-2 and BMP-2 were concentrated using a Filtron Minisette (PallGelman) cross-flow device. pH was adjusted to 5.5, and the material was dialyzed against 0.1 M Tris, 75 mM acetic acid, 0.2 M KH2PO4, 5 mM EDTA, and 6 M urea and then filtered. Subsequently, 150 mg of protein was loaded onto a 5-ml HiTrapTM heparin-Sepharose HP (Amersham Biosciences) column that had been pre-equilibrated with 0.1 M Tris, 125 mM acetic acid, 5 mM EDTA, and 6 M urea (buffer A) containing 0.3 M NaCl. Loading was performed at a flow rate of 4 ml/min. The column was washed with 20 column volumes of buffer A containing 0.6 M NaCl. Elution of dimeric proBMP-2 was finally achieved by application of a linear gradient from 0.6 to 0.8 M NaCl in 5 column volumes. |
| Refolding Assay | Biological assay |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 1 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | Refolding Temperature is not stated |