Refolding Record:
| Protein | |
|---|---|
| Protein Name | Alpha-amylase |
| Abbreviated Name | Alpha-amylase |
| SCOP Family | alpha-Amylases, C-terminal beta-sheet domain |
| Structure Notes | |
| Organism | Pyrococcus woesei |
| UniProt Accession | Q7LYT7 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 435 |
| Molecular Weight | 50184.0 |
| Pi | 4.81736 |
| Molecular Weight | 50184.0 |
| Disulphides | Unknown |
| Full Sequence |
AKYLE LEEGGVIMQA FYWDVPGGGI WWDHIRSKIP EWYEAGISAI WLPPPSKGMS GGYSMGYDPY DYFDLGEYYQ KGTVETRFGS KEELVRLIQT AHAYGIKVIA DVVINHRAGG DLEWNPFVGD YTWTDFSKVA SGKYTANYLD FHPNELHCCD EGTFGGFPDI CHHKEWDQYW LWKSNESYAA YLRSIGFDGW RFDYVKGYGA WVVRDWLNWW GGWAVGEYWD TNVDALLSWA YESGAKVFDF PLYYKMDEAF DNNNIPALVY ALQNGQTVVS RDPFKAVTFV ANHDTDIIWN KYPAYAFILT YEGQPVIFYR DFEEWLNKDK LINLIWIHDH LAGGSTTIVY YDNDELIFVR NGDSRRPGLI TYINLSPNWV GRWVYVPKFA GACIHEYTGN LGGWVDKRVD SSGWVYLEAP PHDPANGYYG YSVWSYCGVG
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Linden A, Niehaus F, Antranikian G (2000) J Chromatography B, 737, 253-259 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | E. coli/H. elongata |
| Expression Strain | BL21(DE3)/ATCC33174 |
| Expression Temp | 37.0 |
| Expression Time | 4h/overnight |
| Expression Vector | pET15b/pHS15 |
| Expression Protocol | E.COLI EXPRESSION: Cells were grown in mineral medium with 20g/L glucose, 10g/L casamino acids, 5mg/L thiamine-HCl. Protein expression was induced in the late logarithmic growth phase with 1mM IPTG. Cells were grown for 4h and then harvested by centrifugation (10min x 7000g). H.ELONGATA EXPRESSION: Cells were grown in 400ml modified SWYE medium in which the final percentage of total salt concentration was decreased to 3%(w/v). Cells were grown overnight with shaking (210rpm) and were harvested by centrifugation (10000g x 10min) |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: hydrophobic interaction chromatography |
| Wash Buffer | 0.05M sodium phosphate pH 7.2, 3mM EDTA, 1%(v/v) Triton X-100 |
| Solubilization Buffer | 0.05M sodium acetate pH 6.0, 3mM EDTA, 20%(v/v) glycerol |
| Refolding Buffer | 0.05M sodium acetate pH 6.0, 50%(v/v) ethylene glycol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 6.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Cells were resuspended in 0.05M sodium acetate pH 6.0 and were lysed by sonication on ice. Lysates were centrifuged (30min x 54000g) and the pellet was washed with 20ml wash buffer. The solution was centrifuged again (1h x 54000g) then the pellet was repeatedly reusupended in solubilisztion buffer followed by stirring at room temperature for 2-4h. The mixture was centrifuged (1h x 54000g) and the superntant (3-10ml sample volumes) was then loaded onto a phenyl superose column previously equilibrated with solubilization buffer. The column was washed with buffer A, then the protein was eluted using with a 20ml increasing linear gradient of refolding buffer. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,Ethylene glycol |
| Additives Concentration | 50% |
| Refolding Yield | 54% |
| Purity | |
| Notes | |