Refolding Record:
| Protein | |
|---|---|
| Protein Name | oxaloacetate decarboxylase, apha-subunit |
| Abbreviated Name | AfOadA |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Archaeoglobus fulgidus |
| UniProt Accession | O29016 |
| 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 | 465 |
| Molecular Weight | 51466.5 |
| Pi | 5.27024 |
| Molecular Weight | 51466.5 |
| Disulphides | 0 |
| Full Sequence |
MRTRDMLPILETLDESGIYSLEVWGGATFDACHRFLNENPWERLREIRKRVKNAKLQMLL
RGQNLVGYRHYPDDIVEKFVQKTIENGLDVFRIFDALNDVRNLISSIKAAKKYNADHIQG
TICYTISPVHTVEKYVEIANELAALEVDSICLKDMAGMLSPKMAYELVKALKKEVGLPIN
VHSHYTSGMASMALLKGVEAGAEMIDTCMSPLSSGTSHPPTESMVYALNELGYDTGVKLD
ILLEAREYFMKIREKYSGYLNPLSTIPDTRVLVYQIPGGMFSNLIAQLQEQNALDRLQEV
LEEVPRVREDLGYPPLVTPTSQIVGVQAVINVLVGERYKVVTRETKDLVKGMYGRTPAPI
KPEIVKKILGDEKPIDCRPADLLEPEFEKRKQELIEAGIENPSDEDVLLYALFPQTGLKF
LKGEMVEEPFPVAGGKIEGSFEVEIDGQKYVVKVAPEK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Dahinden, P., Pos, M. P., Taralczak, M., and Dimroth, P. (2004) Arch Microbiol, 182, 414–420 |
| Project Aim | Structure-Function |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | C43 (DE3) |
| Expression Temp | 25.0 |
| Expression Time | 4 h |
| Expression Vector | pET24b |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | French Press |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 100 mM Tris-HCl, pH 7.5 and 20 mM EDTA |
| Solubilization Buffer | 100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 6 M guanidinium chloride and 100 mM dithiothreitol |
| Refolding Buffer | 100 mM Tris-HCl, pH 8.5, 1 mM EDTA, 1 mM DTT, 0.1% LDAO, and 500 mM L-arginine or 1 M 3-(1-pyridino)-1-propanesulfate (PPS) |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.5 |
| Refolding Temperature | 15.0 |
| Protein Concentration | 50 ug/ml |
| Refolding Time | 10 min |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells containing inclusion bodies were re-suspended in 5 ml per gram of cells 50 mM Tris-HCl, pH 7.0, 1 mM EDTA and 3 mM MgCl2. After the addition of 7.5 mg lysozyme and 50 ug DNase I, the suspension was incubated for 25 minutes at 25°C and then passed three times through a French pressure cell at 110 MPa. Subsequently, 20 mM EDTA, pH 8.0, 0.5 M NaCl and 2% Triton X-100 (final concentration) was added and the mixture incubated for 30 minutes at 4°C. Inclusion bodies were harvested by centrifugation at 20’000x g and washed twice with 100 mM Tris-HCl, pH 7.5 and 20 mM EDTA (10 ml), before they were solubilized with 100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 6 M guanidinium chloride and 100 mM dithiothreitol (at 10 mg protein per ml). After 2-3 h at 25º C, solubilization was complete. After adjusting the pH to 3-4 with 10 mM HCl and centrifugation the supernatant (0.5 ml) was slowly diluted into 50 ml 100 mM Tris-HCl, 1 mM EDTA, 1 mM DTT containing 500 mM L-arginine or 1 M 3-(1-pyridino)-1-propanesulfate (PPS) and/or 0.1% LDAO (adjusted to a final pH of 8.5) at 15°C. After 10 min another 0.5 ml of solubilized protein solution was slowly added. This cycle was repeated 19 times to reach a final guanidinium chloride concentration of just below 1 M. Precipitated protein was removed by passage through a 0.22 um filter. Soluble, but higher aggregates of AfOadA were removed by precipitation with 40% ammonium sulfate. Soluble protein was then precipitated with 60% ammonium sulfate and the precipitate collected by centrifugation. The protein was dissolved in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl and 0.1% LDAO to a concentration of about 1 mg/ml, dialyzed overnight against 2 l of 20 mM Tris-HCl, pH 8.0 containing 100 mM NaCl and subsequently passed through a 0.22 um filter unit. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 24% (of mass) |
| Purity | pure |
| Notes | MGHHHHHHHHHHSSGHIEGRHMAKVKIVDLTLRDGHQSLLATRMRTRDMLPILETLDESGIYSLEVWGGATFDACHRFLNENPWERLREIRKRVKNAKLQMLLRGQNLVGYRHYPDDIVEKFVQKTIENGLDVFRIFDALNDVRNLISSIKAAKKYNADHIQGTICYTISPVHTVEKYVEIANELAALEVDSICLKDMAGMLSPKMAYELVKALKKEVGLPINVHSHYTSGMASMALLKGVEAGAEMIDTCMSPLSSGTSHPPTESMVYALNELGYDTGVKLDILLEAREYFMKIREKYSGYLNPLSTIPDTRVLVYQIPGGMFSNLIAQLQEQNALDRLQEVLEEVPRVREDLGYPPLVTPTSQIVGVQAVINVLVGERYKVVTRETKDLVKGMYGRTPAPIKPEIVKKILGDEKPIDCRPADLLEPEFEKRKQELIEAGIENPSDEDVLLYALFPQTGLKFLKGEMVEEPFPVAGGKIEGSFEVEIDGQKYVVKVAPEKLEDPAANKARKEAELAAATAEQ |