Refolding Record:
| Protein | |
|---|---|
| Protein Name | Translocase of the outer mitochondrial membrane Tom40 |
| Abbreviated Name | TOM40 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Saccharomyces cerevisiae |
| UniProt Accession | P23644 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 409 |
| Molecular Weight | 44288.9 |
| Pi | 5.39 |
| Molecular Weight | 44288.9 |
| Disulphides | 0 |
| Full Sequence |
MSAPTPLAEASQIPTIPALSPLTAKQSKGNFFSSNPISSFVVDTYKQLHSHRQSLELVNP
GTVENLNKEVSRDVFLSQYFFTGLRADLNKAFSMNPAFQTSHTFSIGSQALPKYAFSALF
ANDNLFAQGNIDNDLSVSGRLNYGWDKKNISKVNLQISDGQPTMCQLEQDYQASDFSVNV
KTLNPSFSEKGEFTGVAVASFLQSVTPQLALGLETLYSRTDGSAPGDAGVSYLTRYVSKK
QDWIFSGQLQANGALIASLWRKVAQNVEAGIETTLQAGMVPITDPLMGTPIGIQPTVEGS
TTIGAKYEYRQSVYRGTLDSNGKVACFLERKVLPTLSVLFCGEIDHFKNDTKIGCGLQFE
TAGNQELLMLQQGLDADGNPLQALPQLGRDPAANKARKEAELAAATAEQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Becker, L., Bannwarth, M., Meisinger, C., Hill, K., Model, K., Krimmer, T., Casadio, R., Truscott, K., Schulz, G.E., Pfanner, N., Wagner, R. (2005) J Mol Biol, 353, 1011-1020 |
| Project Aim | Functional Studies |
| Fusion | C-terminal sequence |
| 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 | 6h |
| Expression Vector | pET-9b |
| Expression Protocol | Cells were induced by the addition of IPTG and 1% lactose and incubated for 6hrs prior to harvesting |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6-0.9 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Detergents |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 30mM TrisHCl, pH 8.0, 100mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100 and 2% (w/v) lauryl dimethylamine oxide |
| Solubilization Buffer | 6 M guanidine hydrochloride, 300mM Tris base, 100 mM valine, 50 mM dipotassium hydrogen phosphate, 20 mM beta-mercaptoethanol, 20 mM dodecyldimethyl-a |
| Refolding Buffer | 1.2 M glycerol, 50 mM dipotassium hydrogen phosphate, pH 8.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Inclusion bodies were solubilised in buffer. The solution was dialyzed against refolding buffer. Dialysate was concentrated using a stirred pressure cell, dialyzed again, filtered and loaded onto a 40 ml Q Sepharose FF column equilibrated with 20 mM TrisHCl, pH 9.0, 2 M glycerol, 5 mM polidocanol and 1 mM DTT. The buffer was then changed to 20 mM TrisHCl, pH 8.0, 2 M glycerol, 1 mM dodecylmaltoside, 1 mM DTT and eluted by a gradient to 500 mM NaCl in this buffer. TOM was further purified by gel-permeation chromatography on a Superose 6 column using1.2 glycerol, 20 mM Mops-NaOH, pH 7.0, 1 mM dodecylmaltoside and 150 mM NaCl as buffer. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |