Refolding Record:
| Protein | |
|---|---|
| Protein Name | HIV tat protein |
| Abbreviated Name | HIV tat |
| SCOP Family | Thermolysin-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P04326 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 86 |
| Molecular Weight | 9758.2 |
| Pi | 9.88151 |
| Molecular Weight | 9758.2 |
| Disulphides | 0 |
| Full Sequence |
MEPVDPRLEP WKHPGSQPKT ACTNCYCKKC CFHCQVCFIT KALGISYGRK KRRQRRRAPQ GSQTHQVSLS KQPTSQSRGD PTGPKE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Harper JW and Logsdon NJ (1991) Biochemistry, 30, 8060-8066 |
| 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 | AR120 |
| Expression Temp | 37.0 |
| Expression Time | n/a |
| Expression Vector | not stated |
| Expression Protocol | no details provided |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M urea, 12mM TrisHCl pH 8.0, 10mM DTT |
| Refolding Buffer | 25mM Tris pH 8, 2mM DTT, 1M NaCl, 1mM EDTA |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 16h |
| Redox Agent | DTT |
| Redox Agent Concentration | 2mM |
| Refolding Protocol | Tat-containing cells were sonicated, the protein was then precipitated with poly(ethyleneimine). The poly(ethylenimine) pellet was then solubilized in solubilization buffer and the mixture was centrifuged (20min, 15000g) and the protein was then passed down a CM-sephadex column. The protein was then dialysed for 16h against 1000 volumes of refolding buffer without stirring. The peptides were then purified further by reverse-phase HPLC. |
| Refolding Assay | DNA binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | >95% |
| Notes | |