Refolding Record:
| Protein | |
|---|---|
| Protein Name | Ykt6p SNARE |
| Abbreviated Name | YKT6p |
| SCOP Family | Transforming growth factor (TGF)-beta |
| Structure Notes | |
| Organism | Arabidopsis thaliana |
| UniProt Accession | Q9ZRD6 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 220 |
| Molecular Weight | 24838.1 |
| Pi | 7.76194 |
| Molecular Weight | 24838.1 |
| Disulphides | Unknown |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSHMMKITALLVLK CAPEASDPVI LSNASDVSHF GYFQRSSVKE FVVFVGRTVA SRTPPSQRQS VQHEEYKVHA YNRNGLCAVG FMDDHYPVRS AFSLLNQVLD
EYQKSFGESW RSAKEDSNQP WPYLTEALNK FQDPAEADKL LKIQRELDET KIILHKTIDS VLARGEKLDS LVEKSSDLSM ASQMFYKQAK KTNSCCTIL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Vincent P, Dieryck W, Maneta-Peyret L, Moreau P, Cassagne C, Santarelli X (2004) J Chromatography B, 808, 83-89 |
| Project Aim | Functional Studies |
| 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 | 5h |
| Expression Vector | pET15b |
| Expression Protocol | LB medium with 0.1mg/ml ampicillin was inoculated with a 1/200 overnight seed culture. Protein expression was induced with 0.3mM IPTG. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD 600 = 0.7 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | 2M urea, 20mM TrisHCl, 0.5M NaCl, 2% Triton X-100 pH 8.0 |
| Solubilization Buffer | 20mM TrisHCl, 0.5M NaCl, 5mM imidazole, 6M GdnHCl, 1mM 2-mercaptoethanol pH 8.0 |
| Refolding Buffer | 20mM TrisHCl, 0.5M NaCl, 20mM imidazole, 1mM 2-mercaptoethanol pH 8.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 1mM,1mM |
| Refolding Protocol | After expression, cells were harvested by centrifugation and sonicated for 3x3sec with 2min between pulses. The mixture was then centfrifuged (25000g x 30min, 4degC) and the pellet was resuspended in 40ml cold wash buffer and sonicated again. Following centrifugation (25000g x 30min, 4degC), pellets were subjected again to another urea wash. Following resuspension in 5ml solubilization buffer, the mixture was centrifuged at high speed at 4degC and the supernatant was filtered through a 0.22micron filter. The supernatant was then loaded ont a nickel-chelating sepharose column equilibrated with solubilization buffer. The column was then washed with 80ml of solubilization buffer, followed by a further 5ml wash with 20mM TrisHCl, 0.5M NaCl, 20mM imidazole, 6M urea, 1mM 2-mercaptoethanol pH 8.0. The protein was then refolded by a 30ml 6-0M urea gradient in 20mM TrisHCl, 0.5M NaCl, 20mM imidazole, 1mM 2-mercaptoethanol pH 8.0, and the column was subsequently washed with 7ml refolding buffer. The protein was then eluted in 20mM TrisHCl, 0.5M NaCl, 1mM 2-mercaptoethanol pH 8.0 with a linear gradient of 20-500mM imidazole. Selected fractions were pooled and passed through a Sephadex G25 column. |
| Refolding Assay | Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 88% |
| Purity | >94% |
| Notes | |