Refolding Record:
| Protein | |
|---|---|
| Protein Name | Casbene synthase |
| Abbreviated Name | Casbene |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Ricinus communis |
| UniProt Accession | P59287 |
| 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 | 611 |
| Molecular Weight | 68965.6 |
| Pi | 5.34864 |
| Molecular Weight | 68965.6 |
| Disulphides | 0 |
| Full Sequence |
MALPSAAMQSNPEKLNLFHRLSSLPTTSLEYGNNRFPFFSSSAKSHFKKPTQACLSSTTH
QEVRPLAYFPPTVWGNRFASLTFNPSEFESYDERVIVLKKKVKDILISSTSDSVETVILI
DLLCRLGVSYHFENDIEELLSKIFNSQPDLVDEKECDLYTAAIVFRVFRQHGFKMSSDVF
SKFKDSDGKFKESLRGDAKGMLSLFEASHLSVHGEDILEEAFAFTKDYLQSSAVELFPNL
KRHITNALEQPFHSGVPRLEARKFIDLYEADIECRNETLLEFAKLDYNRVQLLHQQELCQ
FSKWWKDLNLASDIPYARDRMAEIFFWAVAMYFEPDYAHTRMIIAKVVLLISLIDDTIDA
YATMEETHILAEAVARWDMSCLEKLPDYMKVIYKLLLNTFSEFEKELTAEGKSYSVKYGR
EAFQELVRGYYLEAVWRDEGKIPSFDDYLYNGSMTTGLPLVSTASFMGVQEITGLNEFQW
LETNPKLSYASGAFIRLVNDLTSHVTEQQRGHVASCIDCYMNQHGVSKDEAVKILQKMAT
DCWKEINEECMRQSQVSVGHLMRIVNLARLTDVSYKYGDGYTDSQQLKQFVKGLFVDPIS
I
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hill AM, Cane DE, Mau CJ, West CA. (1996) Archives Biochemistry and Biophysics, 336, 283-289 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pSM102 |
| Expression Temp | 37.0 |
| Expression Time | 2h |
| Expression Vector | pET21d |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 10% glycerol, 50mM Tris-HCl, 1mM EDTA, 10mM betamercaptoethanol, 2mg.ml lysozyme, 1mg.ml deoxycholate, pH 8.3 |
| Solubilization Buffer | 10mM NaOH |
| Refolding Buffer | 20% glycerol, 50mM Tris-HCl, 10mM betamercaptoethanol, 1mM EDTA |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.03mg/ml |
| Refolding Time | 2h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | n/a |
| Refolding Protocol | 20ml of the wash buffer containing the target protein was diluted 10 fold into 10mM NaOH and was stirred for 1h hour at 0C. Insoluble material was removed with centrifugation and the supernantant was diluted 80 fold with refolding buffer. The solution was stirred slowly for 2h at 4C and subsequently purified by Q sepharose chromatography. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 17mg/L culture |
| Purity | single band on SDS PAGE |
| Notes | |