Refolding Record:
| Protein | |
|---|---|
| Protein Name | 1-aminocyclopropane-1-carboxylate synthase |
| Abbreviated Name | ACC synthase |
| SCOP Family | GABA-aminotransferase-like |
| Structure Notes | |
| Organism | Cucurbita pepo (Squash) |
| UniProt Accession | P23279 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 503 |
| Molecular Weight | 57033.9 |
| Pi | 7.10334 |
| Molecular Weight | 57033.9 |
| Disulphides | 0 |
| Full Sequence |
MRGSHHHHHH MGFHQIDERN QALLSKIALD DGHGENSPYF DGWKAYDNDP FHPENNPLGV IQMGLAENQL SFDMIVDWIR KHPEASICTP EGLERFKSIA NFQDYHGLPE FRNAIANFMG KVRGGRVKFD PSRIVMGGGA TGASETVIFC LADPGDAFLV
PSPYYAGFDR DLKWRTRAQI IRVHCNGSNN FQVTKAALEI AYKKAQEANM KVKGVIITNP SNPLGTTYDR DTLKTLVTFV NQHDIHLICD EIYSATVFKA PTFTSIAEIV EQMEHCKKEL IHILYSLSKD MGLPGFRVGI IYSYNDVVVR RARQMSSFGL VSSQTQHLLA AMLSDEDFVD KFLAENSKRV GERHARFTKE
LDKMGITCLN SNAGVFVWMD LRRLLKDQTF KAEMELWRVI INEVKLNVSP GSSFHVTEPG WFRVCFANMD DNTVDVALNR IHSFVENIDK KEDNTVAMPS KTRHRDNKLR LSFSFSGRRY DEGNVLNSPH TMSPHSPLVI AKN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Huxtable S, Zhou H, Wong S, Li N. (1998) Protein Expression and Purification, 12, 305-314 |
| Project Aim | Structure-Function |
| 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 |
| Expression Temp | 37.0 |
| Expression Time | overnight |
| Expression Vector | pET30a |
| Expression Protocol | 800ml of LBM9 medium was inoculated with overnight culture at 37degC OD600 reached 0.6. 1mM IPTG and 5microM ACC synthase were then added and cell cultures were allowed to grow for l h at 25degC before 200 microg/ml rifampicin was added. Incubation was continued for an additional 2h at 25degC, then cells were harvested by centrifugation. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 100mM EPPS, 5mM EDTA, 10mM DTT, 20 microM PLP, 1mM PMSF, 0.1% Triton X-100 pH 8 |
| Solubilization Buffer | 100mM MOPS, 20mM Na2CO3, 6M urea, 10mM EDTA, 50mM DTT, 50 microM PLP, 0.1mM PMSF pH 9.5 |
| Refolding Buffer | 100mM MOPS, 5mM DTT, 33mM Chaps, 1% glycerol, 5mM GSH, 50 microM PLP, 0.1mM PMSF |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.045 mg/ml |
| Refolding Time | 60h |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a,n/a,5mM |
| Refolding Protocol | The inclusion bodies were washed once with 100mM EPPS, 5mM EDTA, 10mM DTT, 20 microM PLP, 1mM PMSF, 0.1% Triton X-100 pH 8 and subsequently with the same buffer containing 0.5M urea but not Triton. The protein was solubilised with 100mM MOPS, 20mM Na2CO3, 6M urea, 10mM EDTA, 50mM DTT, 50 microM PLP, 0.1mM PMSF pH 9.5 and refolding by dilution with 100mM MOPS, 5mM DTT, 33mM Chaps, 1% glycerol, 5mM GSH, 50 microM PLP, 0.1mM PMSF such that the final urea concentration was 1.2M. The protein was then dialysed against the same refolding buffer containing 1M urea for 12h. The refolding buffer was replaced without urea and dialysis continued for a further 18h. The buffer was then exchanged every 6h for 30h. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 1% |
| Refolding Yield | 32% |
| Purity | few contaminating bands on SDS PAGE |
| Notes | |