Refolding Record:
| Protein | |
|---|---|
| Protein Name | DevS Histidine Protein Kinase |
| Abbreviated Name | DevS |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Mycobacterium tuberculosis |
| UniProt Accession | Q7TX69 |
| 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 | 587 |
| Molecular Weight | 62228.6 |
| Pi | 4.90687 |
| Molecular Weight | 62228.6 |
| Disulphides | Unknown |
| Full Sequence |
MTTGGLVDENDGAAMRPLRHTLSQLRLHELLVEVQDRVEQIVEGRDRLDGLVEAMLVVTA
GLDLEATLRAIVHSATSLVDARYGAMEVHDRQHRVLHFVYEGIDEETVRRIGHLPKGLGV
IGLLIEDPKPLRLDDVSAHPASIGFPPYHPPMRTFLGVPVRVRDESFGTLYLTDKTNGQP
FSDDDEVLVQALAAAAGIAVANARLYQQAKARQSWIEATRDIATELLSGTEPATVFRLVA
AEALKLTAADAALVAVPVDEDMPAADVGELLVIETVGSAVASTVGRTIPVAGAVLREVFV
NGIPRRVDRVDLEGLDELADAGPALLLPLRARGTVAGVVVVLSQGGPGAFTDEQLEMMAA
FADQAALAWQLATSQRRMRELDVLTDRDRIARDLHDHVIQRLFAIGLALQGAVPHERNPE
VQQRLSDVVDDLQDVIQEIRTTIYDLHGASQGITRLRQRIDAAVAQFADSGLRTSVQFVG
PLSVVDSALADQAEAVVREAVSNAVRHAKASTLTVRVKVDDDLCIEVTDNGRGLPDEFTG
SGLTNLRQRAEQAGGEFTLASVPGASGTVLRWSAPLSQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Saini DK, Pant N, Das TK, Tyagi JS. (2002) Protein Expression and Purification, 25, 203-208 |
| Project Aim | Structural 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 | pPROEx-HTc |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | unknown |
| Solubilization Buffer | 20mM Tris-HCl, 500mM NaCl, 10% glycerol, 8M urea, pH 8.0 |
| Refolding Buffer | 20mM Tris-HCl, 500mM NaCl, 10% glycerol, 20mM imidazole, 0.5mM GSSG, 5mM GSH |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 16.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The whole cell pellet was resuspended in 10 ml of solubilization/denaturation buffer (20 mM Tris HCl, pH 8.0, 500 mM NaCl, 10% glycerol containing 8 M urea) and incubated 37C for 2 h with shaking, followed by centrifugation at 12,000g for 15 min to remove the cell debris other insoluble material. The supernatant fraction taining the solubilized inclusion bodies was processed for protein purification. Gm HCl at 6 M could also used in place of urea with identical results. The denatured proteins were loaded onto a 2-ml column of preequilibrated Ni+2?NTA agarose (Qiagen, GmbH, Germany) and incubated for 2 h at room temperature. The column was shifted to 168C and the unbound protein was collected as flowthrough. The column was washed with 3 bed volumes of denaturation buffer containing 20mM imidazole and then washed stepwise with decreasing concentrations of urea (6 to 1 M) in refolding buffer (20 mM Tris HCl, pH 8.0, 500 mM NaCl, 10% glycerol, 20 mM imidazole, 0.5 mM oxidized glutathione (GSSG) and 5mMreduced glutathione (GSH)). The column was then washed with 2 bed volumes of refolding buffer without urea to remove contaminating E. coli proteins. The bound protein was eluted with 5ml refolding buffer containing 250 mM imidazole in fractions of 1 ml. |
| Refolding Assay | Phosphorylation |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 40mg/L culture |
| Purity | |
| Notes | |