Refolding Record:
| Protein | |
|---|---|
| Protein Name | ATP synthase subunit alpha |
| Abbreviated Name | RrF1 alpha |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Rhodospirillum rubrum |
| UniProt Accession | P05036 |
| 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 | 511 |
| Molecular Weight | 55026.2 |
| Pi | 5.84 |
| Molecular Weight | 55026.2 |
| Disulphides | Unknown |
| Full Sequence |
MEIRAAEISAILKEQIANFGTEAESAEVGQVLSVGDGIARVYGLDNVQAGEMVEFANGVKGMALNLESDNVGIVIFGEDRGIKEGDVVKRTQTIVDVPVGKGLLGRVVDGLGNPIDGKGDLVDVERKRAEVKAPGIIPRKSVHEPVQTGIKAIDSLIPIGRGQRELIIGDRQTGKTAVILDTILNQKAVNDKAKDDSEKLFCVYVAVGQKRSTVAQVVKVLADHGALDYTIVVAATASEPAPLQFLAPYTGCTMGEFFRDNGMHAVIFYDDLTKQAVAYRQMSLLLRRPPGREAFPGDVF
YLHSRLLERAAKLNDDNGAGSLTALPVIETQANDVSAYIPTNVISITDGQIFLETDLFFKGIRPAVNVGLSVSRVGSSAQIKAMKQVAGSIKLELAQYREMAAFAQFASDLDPATQKLLARGARLTELLKQAQYSPLAVEEQVCVIYAGTRGYLDKLKTTDVVRYEASLLGALRTSGADLLESIRTGKALSKEIEQKLVKFLDDFGKKFA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Du Z, Gromet-Elhanan Z. (1999) Eur J Biochem., 263, 430-437 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DK8 |
| Expression Temp | 37.0 |
| Expression Time | overnight |
| Expression Vector | pTZ18 |
| Expression Protocol | The RrF1α gene was amplified from R. rubrum genomic DNA by PCR, cloned, fully sequenced as described in Du and Gromet-Elhanan [ 30], and found to be identical to the earlier published sequence [ 31]. It was ligated into the expression vector pTZ18 [ 32], and the recombinant plasmid, designated pTZα, was transformed into the unc operon-deleted E. coli DK8 strain [ 33] and grown overnight at 37 °C in Luria–Bertani medium containing ampicillin at 100 µg·mL−1. The expressed RrF1α subunit appeared exclusively in insoluble inclusion bodies, which were isolated from washed cells according to the method of Du and Gromet Elhanan [ 30] with some modification. The cells were resuspended in TE buffer containing: 50 m m Tris/HCl, pH 8.0, and 2 m m EDTA, together with the following protease inhibitors: phenylmethanesulfonyl fluoride, benzamidine and Nαp-tosyl- l-lysine chloromethyl ketone at 1 m m, 2 m m and 10 µg·mL−1, respectively, and disrupted by sonication. The inclusion bodies were sedimented at 10 000 g for 20 min, washed three times with TE buffer containing the protease inhibitors, and stored at −80 °C. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | TE buffer (50 m m Tris/HCl, pH 8.0, and 2 m m EDTA) |
| Solubilization Buffer | 1 h at 4 °C in TE buffer with 50 mM dithiothreitol, 5 M urea and the protease inhibitors (phenylmethanesulfonyl fluoride, benzamidine and Nαp-to |
| Refolding Buffer | 50 mM Tris/HCl, pH 8.0, 10 mM dithiothreitol, 20% glycerol, 5 M urea, the protease inhibitors and 50 mM of MgCl2 and ATP |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 10 mM |
| Refolding Protocol | The washed inclusion bodies were solubilized by incubation for 1 h at 4 °C in TE buffer containing also 50 m m dithiothreitol, 5 m urea and the protease inhibitors. The remaining insoluble material was removed by centrifugation at 20 000 g for 30 min. The supernatant containing almost pure RrF1α[ 30] was diluted to 50 µg·mL−1, unless otherwise indicated, in the refolding buffer containing: 50 m m Tris/HCl, pH 8.0, 10 m m dithiothreitol, 20% glycerol, 5 m urea, the protease inhibitors and 50 m m of MgCl2 and ATP. Each sample was dialyzed overnight at 4 °C against 50 vol. of TG buffer containing: 50 m m Tris/HCl, pH 8.0, and 20% glycerol. The dialyzed samples were concentrated to ≈ 5 mg·mL−1 by Centriprep-10 (Amicon) and cleared by centrifugation at 20000 g for 1 h. The stable refolded RrF1α was freed from residual MgATP by elution-centrifugation through Sephadex G-50 columns [ 34], equilibrated with TGN buffer containing: 50 m m Tricine/NaOH, pH 8.0, 20% glycerol and 50 m m NaCl, and stored at −80 °C. |
| Refolding Assay | Assays of refolding efficiency |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |