Refolding Record:
| Protein | |
|---|---|
| Protein Name | Myoglobin |
| Abbreviated Name | Mb |
| SCOP Family | Globins |
| Structure Notes | |
| Organism | Physeter catodon |
| UniProt Accession | P02185 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 155 |
| Molecular Weight | 17199.9 |
| Pi | 8.70957 |
| Molecular Weight | 17199.9 |
| Disulphides | 0 |
| Full Sequence |
VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASE
DLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRH
PGDFGADAQGAMNKALELFRKDIAAKYKELGYQG
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Ribeiro-Júnior, E.A., Regis, W.C.B., Tasic, L., and Ramos, C.H.I. (2003) Protein Expression and Purification, 28, 202-208 |
| Project Aim | Biophysical Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 42.0 |
| Expression Time | 4h |
| Expression Vector | pET |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication/French Press |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | Tris-HCl 100 mM, KCl 100 mM, EDTA 1 mM pH 8.0 |
| Solubilization Buffer | Tris-HCl 100 mM pH 8.0, Gdm-Cl 8 M |
| Refolding Buffer | Sodium phosphate 50 mM pH 7.2 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 24hs |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Expression and purification. LB media containing carbenicillin (Pharmacia) was inoculated with an overnight transformed culture of BL21(DE3) E. coli strand (Novagen). The culture was grown at 37 „aC until it reached an OD600 of between 0.8 and 1.0 U.A., when IPTG was added to a final concentration of 0.4 mM (15). The temperature was either maintained at 37 „aC, for conventional protein expression, or changed to 42 „aC for expression in inclusion bodies. After 5 hours, the bacteria culture was sedimented by centrifugation, ressuspended in 20 ml/L of Lysis Buffer, and passed three times throughout the French„µ press at 2000 psi. The DEAE column was equilibrated with 5 bed volumes of equilibration buffer and the protein was recovered in the flow throughout. The CM column was equilibrated with 5 bed volumes of equilibration buffer but the protein was recovered with increasing concentrations of NaCl (0 to 100 mM) in equilibration buffer (10 bed volumes). The examination of expression yield and protein profile purification was performed by SDS polyacrylamide gel (SDS-PAGE) as described by Laemmli (17). The composition of working buffers follows. Lysis Buffer: Tris-HCl 100 mM, KCl 100 mM, EDTA 1 mM pH 8.0. Solubilization Buffer: Tris-HCl 100 mM pH 8.0, Gdm-Cl 8 M. Equilibration Buffer: Sodium phosphate 50 mM pH 7.2. No protease inhibitors were included in the buffers. [See manuscript for details.] |
| Refolding Assay | 1H chemical shift (ppm) |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | >90% |
| Purity | >95% |
| Notes | |