Refolding Record:
| Protein | |
|---|---|
| Protein Name | Aminoglycoside nucleotidyltransferase(2")-Ia |
| Abbreviated Name | ANT2" |
| SCOP Family | nucleotidyltransferases |
| Structure Notes | |
| Organism | Pseudomonas aeruginosa |
| UniProt Accession | Q6X3H6 |
| 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 | 219 |
| Molecular Weight | 24162.4 |
| Pi | 4.96 |
| Molecular Weight | 24162.4 |
| Disulphides | 1 |
| Full Sequence |
MPRASKQQARYAVGRCLMLWSSNDVTQQGSRPKTKLGRMDTTQVTLIHKILAAADERN
LPLWIGGGWAIDARLGRVTRKHDDIDLTFPGERRGELEAIVEMLGGRVMEELDYGFLAE
IGDELLDCEPAWWADEAYEIAEAPQGSCPEAAEGVIAGRPVRCNSWEAIIWDYFYYADE
VPPVDWPTKHIESYRLACTSLGAEKVEVLRAAFRSRYAA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Wright, E., and Serpersu, E. H. (2004) Protein Expression and Purification, 35, 373-380 |
| Project Aim | Biophysical 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 | 6 hours |
| Expression Vector | pET 22b(+) |
| Expression Protocol | Cells are grown in LB media containing amphicillin. Culture is induced when the OD reaches to 600. Six hours after cells were harvested and lysed. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = n/a |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 10 mM Tris-HCl pH 8.0, o.1 M NaCl, 1 mM EDTA |
| Solubilization Buffer | 8M urea, 0.1 M Tris-HCl pH 8.0, 10 mM EDTA |
| Refolding Buffer | 0.1 M Tris-HCl pH 8.5, 0.2 M KCl, 0.4 M Arginine, 2% glycine, 10 mM GSH, 1 mM GSSG |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | <50 ug/ml |
| Refolding Time | 8h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 10 mM GSH, 1 mM GSSG,10 mM GSH, 1 mM GSSG,10 mM GSH, 1 mM GSSG |
| Refolding Protocol | To 10ml of each refolding buVer were added 0.25mg (about 10 l) of inclusion bodies solubilized in 8M urea, 0.1M Tris–HCl, pH 8.0, and 10mM DTT. After 8 h, the solution was concentrated to 2ml under nitrogen in a stirred cell ultraWltration unit with a modiWed polyethersulfone membrane (Pall Gelman). |
| Refolding Assay | Enzyme activity,HPLC |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine,Glycine |
| Additives Concentration | 2% glycine, 0.4 M L-arginine |
| Refolding Yield | 96% |
| Purity | >95 |
| Notes | n/a |