Refolding Record:
| Protein | |
|---|---|
| Protein Name | Alpha-tocopherol transfer protein |
| Abbreviated Name | alpha-TTP |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P49638 |
| 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 | 318 |
| Molecular Weight | 36124.5 |
| Pi | 8.46882 |
| Molecular Weight | 36124.5 |
| Disulphides | 0 |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFELRR MAEARSQPSA GPQLNALPDH SPLLQPGLAA LRRRAREAGV PLAPLPLTDS FLLRFLRARD FDLDLAWRLL KNYYKWRAEC PEISADLHPR SIIGLLKAGY HGVLRSRDPT GSKVLIYRIA HWDPKVFTAY DVFRVSLITS ELIVQEVETQ RNGIKAIFDL EGWQFSHAFQ ITPSVAKKIA AVLTDSFPLK VRGIHLINEP
VIFHAVFSMI KPFLTEKIKE RIHMHGNNYK QSLLQHFPDI LPLEYGGEEF SMEDICQEWT NFIMKSEDYL SSISESIQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Panagabko C, Morley S, Neely S, Lei H, Manor D, Atkinson J (2002) Protein Expression and Purification, 24, 395-403 |
| Project Aim | Structure-Function |
| Fusion | N-terminal hexahistidine tag and Protein S peptide 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 | 3-4h |
| Expression Vector | pET30 |
| Expression Protocol | Cells were grown until OD600 reached 0.6, at which point 1mM IPTG was added. Cells were then grown a further 3-4h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | 0.05M phosphate, 1% Triton X-100 |
| Solubilization Buffer | PBS, 8M urea, 0.06M imidazole |
| Refolding Buffer | 20mM phosphate pH 7.6, 0.5M arginine |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.6 |
| Refolding Temperature | 14.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cell pellet was resuspended in PBS, and lysozyme was added to a concentration of 100 mg/mL. The sample was incubated at room temperature for 15 min and then sonicated on ice. The soluble fraction was obtained from the supernatant after centrifugation and the re- maining insoluble matter was washed twice with 0.05 M phosphate, pH 7.6, containing either 1.0 % Triton X-100 or 1 M urea. Following centrifugation for 25 min at 43,000g and 4C, the pellet was solubilized in PBS containing 8Murea and 0.06Mimidazole (start buffer). The insoluble fraction was obtained following incubation at room temperature for 20 min followed by centrifugation. In order to refold the protein while still bound to the metal affinity column, a second wash was implemented prior to elution. After the column was washed with start buffer, a second wash of 0.5 M arginine, 25 mM phosphate, pH 7.6, was used to establish refolding conditions on the column. At a scale of about 8 mg of total resolubilized protein having a volume of 4 mL, 8?2 mL of refolding buffer was passed through the column at 2 mL/min. Elution was then performed immediately with 400 mM imidazole in PBS also containing 0.5 M arginine. Aliquots for analysis were desalted by ultrafiltration using Ultrafree Biomax-10K centrifugal devices from Millipore (Bedford, MA). |
| Refolding Assay | Alpha-Tocopherol binding assay |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.5M |
| Refolding Yield | 20.8mg/1L culture |
| Purity | Not stated |
| Notes | |