Refolding Record:
| Protein | |
|---|---|
| Protein Name | Human T-cell leukemia virus type I protease |
| Abbreviated Name | HTLV-1 protease |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | HTLV |
| UniProt Accession | P10274 |
| 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 | 151 |
| Molecular Weight | 16600.0 |
| Pi | 6.68699 |
| Molecular Weight | 16600.0 |
| Disulphides | 0 |
| Full Sequence |
MGHHHHHHHHHH SSGHIDDDKHMLED PVIPLDPA RRPVIKAQVD
TQTSHPKTIE ALLDTGADMT VLPIALFSSN TPLKNTSVLG AGGQTQDHFK LTSLPVLIRL PFRTTPIVLT SCLVDTKNNW AIIGRDALQQ CQGVLYLPEA KGPPVIL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Ding YS, Rich DH, Ikeda RA (1998) Biochemistry, 37, 17514-17518 |
| 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(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pET19b |
| Expression Protocol | 30ml cultures of E.coli were grown at 37degC in LB medium until OD600 reached 0.6, at which point expression was induced by the addition of 0.4mM IPTG. Cells were grown for a further 3h and then harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 20mM Tris pH 7.9, 5mM imidazole, 500mM NaCl, 8M urea |
| Refolding Buffer | 1: 10mM sodium acetate pH 3.5, 2: 100mM sodium citrate pH 5.3, 5mM EDTA, 1mM DTT, 1M NaCl |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | yes |
| Refolding pH | 3.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 8h |
| Redox Agent | DTT |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | Cells were resuspended in 20mM Tris pH 7.9, 5mM imidazole, 500mM NaCl and sonicated. The bacterial lysate was cleared by centrifugation and the pellet was resuspended in solubilization buffer. The mixture was centrifuged and the supernatant was then loaded onto a 1ml His-bind column. The column was washed with solubilization buffer firstly without imidazole, then with 20mM imidazole. The protein was eluted with solubilization buffer containing 1M imidazole. The purified protein was refolded by sequencial dialysis against refolding buffers 1 and 2. Autoprocessing of the histidine-tag occured during dialysis against refolding buffer 2. The processed protein coprecipitated with unprocessed protein. Following dialysis, the precipitated protein was collected by centrifugation (20000g, 20min, 40degC), redisolved in solubilization buffer adn reloaded onto a second His-Bind column. The mature protease eluted immediately (ie. did not bind to the column), and was collected. These fractions were pooled, dialyzed against Refolding buffer 1 containing 1mM DTT and stored at 40degC. |
| Refolding Assay | Ultraviolet (UV) Absorbance |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 100microg/30ml culture |
| Purity | |
| Notes | |