Refolding Record:
| Protein | |
|---|---|
| Protein Name | VirG |
| Abbreviated Name | n/a |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Agrobacterium tumefaciens |
| UniProt Accession | Q8VT91 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 241 |
| Molecular Weight | 27109.3 |
| Pi | 6.93 |
| Molecular Weight | 27109.3 |
| Disulphides | Unknown |
| Full Sequence |
MKHILVIDDDAAMRHLIKEYLTVHALRVTAVSDSQQFTRVLASETVDVVVVDLNLGREDGLEIVRSLATKSDVPIIIISGDRLDEADKVVALELGASDFIAKPFGTREFLARIRVALRERPNVMRTKDRRTFCFAGWTLSLRQRRLTSTQGGDVKLTAGEFNLLVAFLEKPRDVLSREQLLIASRVREEEVYDRSIDVLILRLRRKLEEDAANPHLIKTARGAGYFFDADVNISFGGMMAA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Jin SG, Roitsch T, Christie PJ, Nester EW. (1990) J Bacteriol., 172, 531-537 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DH5α |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pPC401 |
| Expression Protocol | VirG protein purification and renaturation. E. coli DHcS(pPC401) was induced with 1 mM isopropyl-p-D-thio-galactopyranoside, for 3 h, and the insoluble pellet fraction was used to purify the VirG protein by the method of Hager and Burgess (11) |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | n/a |
| Solubilization Buffer | 50 mM Tris hydrochloride [pH 8.0], 1 mM EDTA, 5 mM dithiothreitol, 6 M guanidine hydrochloride |
| Refolding Buffer | 20 mM HEPES [N-hydroxyethylpiperazine-N\'-2-ethanesulfonic acid; pH 7.4], 50 mM KCl, 5 mM dithiothreitol, 50% glycerol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.4 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 18 h |
| Redox Agent | DTT |
| Redox Agent Concentration | 5 mM |
| Refolding Protocol | The purified protein was suspended in buffer D (50 mM Tris hydrochloride [pH 8.0], 1 mM EDTA, 5 mM dithiothreitol, 6 M guanidine hydrochloride) to a final concentration of 0.05 mg/ml. This solution was allowed to dialyze against 200 volumes of renaturation buffer R (20 mM HEPES [N-hydroxyethylpiperazine-N\'-2-ethanesulfonic acid; pH 7.4], 50 mM KCl, 5 mM dithiothreitol, 50% glycerol) for 18 h at 4°C without stirring and then for an additional 18 h in fresh renaturation buffer with stirring. The renatured protein was stored at -20°C. |
| Refolding Assay | Western Blot,Binding assay |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 50% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |