Refolding Record:
| Protein | |
|---|---|
| Protein Name | 4-aminobutyrate aminotransferase, mitochondrial also known as GABA transaminase |
| Abbreviated Name | n/a |
| SCOP Family | GABA-aminotransferase-like |
| Structure Notes | |
| Organism | Pig (Sus scrofa) |
| UniProt Accession | P80147 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 473 |
| Molecular Weight | 53297.1 |
| Pi | 7.19 |
| Molecular Weight | 53297.1 |
| Disulphides | Unknown |
| Full Sequence |
SQAAAKVDVEFDYDGPLMKTEVPGPRSRELMKQLNIIQNAEAVHFFCNYEESRGNYLVDVDGNRMLDLYSQISSIPIGYSHPALVKLVQQPQNVSTFINRPALGILPPENFVEKLRESLLSVAPKGMSQLITMACGSCSNENAFKTIFMWYRSKERGQSAFSKEELETCMINQAPGCPDYSILSFMGAFHGRTMGCLATTHSKAIHKIDIPSFDWPIAPFPRLKYPLEEFVKENQQEEARCLEEVEDLIVKYRKKKKTVAGIIVEPIQSEGGDNHASDDFFRKLRDISRKHGCAFLVDEVQTGGGSTGKFWAHEHWGLDDPADVMTFSKKMMTGGFFHKEEFRPNAPYRIFNTWLGDPSKNLLLAEVINIIKREDLLSNAAHAGKVLLTGLLDLQARYPQFISRVRGRGTFCSFDTPDESIRNKLISIARNKGVMLGGCGDKSIRFRPTLVFRDHHAHLFLNIFSDILADFK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kim YT, Song YH, Churchich JE. (1997) Biochemica et Biophysica Acta, 1337, 248-56 |
| Project Aim | Expression and identification |
| Fusion | T7 |
| 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 h |
| Expression Vector | pYKG-1 |
| Expression Protocol | Recombinant pig 4-aminobutyrate aminotransferase was overproduced using T7 RNA polymerase expression system [15, 16]. To express the recombinant 4-aminobutyrate aminotransferase genes in vivo, pYKG-1 and pYKG-2 plasmids were transformed into the E. coli BL21(DE3). Cells were grown in LB broth containing 50 μg/ml of ampicillin and 25 μg/ml of chloramphenicol at 37°C. At a cell density corresponding to A590=0.6, isopropyl β-thiogalactopyranoside (IPTG) was added at a final concentration of 0.4 mM to induce the expression of T7 RNA polymerase [16]. After induction, the cells were incubated for three additional hours and then harvested by centrifugation at 8000×g for 15 min in a Sorvall GS-3 rotor. The cell paste was resuspended in 0.01 M potassium phosphate (pH 7.4), 25 mM ethylenediaminetetraacetic acid (EDTA), and 10% sucrose, and again harvested by centrifugation. The cell paste (20 g) was resuspended in 90 ml of 0.01 M potassium phosphate (pH 7.4), 1 mM EDTA, and 10% sucrose, and was frozen in liquid N2 and stored at −80°C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 590 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50 ml of 0.05 M potassium phosphate (pH 7.4), containing 1 mM DTT, and 7.2 M urea |
| Solubilization Buffer | 20 ml of 0.01 M potassium phosphate (pH 7.4), 1 mM DTT, and 7.2 M urea |
| Refolding Buffer | 0.01 M potassium phosphate (pH 7.4) containing 1 mM DTT and 4 M urea (see notes) |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.4 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 8 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Purified recombinant 4-aminobutyrate aminotransferase was renatured by a sequential dialysis method. The samples were first dialyzed against 0.01 M potassium phosphate (pH 7.4) containing 1 mM DTT and 4 M urea for at least 8 h at 4°C. The next three dialysis steps employed the same potassium phosphate buffer (pH 7.4) containing 1 mM DTT and the following urea concentrations. The second contained 2M urea, whereas the third contained 1 M urea. The final buffer contained 0.01 M potassium phosphate (pH 7.4), containing 1 mM EDTA, 1 mM -ketoglutarate, 10% glycerol. The final dialysis was stopped after 4 h and 10 μM of pyridoxal 5′-P was added to the dialysate. These samples were then assayed for aminotransferase activity. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 10% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | The refolding method is sequential dialysis and there are 4 steps. Each steps has different buffer (see refolding protocol) |