Refolding Record:
| Protein | |
|---|---|
| Protein Name | RV1120c (hypothetical protein) |
| Abbreviated Name | RV1120c |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Mycobacterium tuberculosis |
| UniProt Accession | O06572 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 162 |
| Molecular Weight | 17816.2 |
| Pi | 8.98226 |
| Molecular Weight | 17816.2 |
| Disulphides | Unknown |
| Full Sequence |
SGGREAVK TVWQTANLVR KEGFGAAVRS SIEDPADWAE VERPDLARVT PDGRVVILFS DIEESTALDE RIGDRTWVKL IGAHDKLVHE LVRRWSGHMV TSQGDGFMIA FARAEQAVRC GIDIQDALRN SAKRKRNQGI RVRIGTTWGA RCGTVTICSA ATSQ
|
| Notes | unclear as to whether there are flanking sequences from the vector |
| Expression | |
|---|---|
| Report | Shenoy AR, Srinivas A, Mahalingam M, Visweswariah SS (2005) Biochimie, 87, 557-563 |
| 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 | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3-12h |
| Expression Vector | pRSET |
| Expression Protocol | Cells were induced with IPTG and incubated at temperatures ranging from 18 to 37degC for 3-12h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20mM TrisHCl pH 8.2, 2mM 2-mercaptoethanol, 1% Triton-X100 |
| Solubilization Buffer | 20mM TrisHCl pH 8.2, 2mM DTT, 8M urea |
| Refolding Buffer | 20mM TrisHCl pH 8.2, 10mM 2-mercaptoethanol, 10% glycerol |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.2 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 10mM,10mM |
| Refolding Protocol | Cells were lysed in 50mM TrisHCl pH 8.2, 5mM 2-mercaptoethanol, 100mM NaCl, 2mM MPSF and centrifuged (3000g, 30min). The inclusion body pellet was washed in wash buffer, once with Triton-X100, followed by washes without Triton-X100. The inclusion bodies were then solubilized in solubilization buffer, then purified under denaturing conditions containing 8M urea using Ni-NTA resin. The purified protein was then dialyzed against refolding buffer. |
| Refolding Assay | Enzyme activity,Western Blot,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 10% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |