Refolding Record:
| Protein | |
|---|---|
| Protein Name | Agmatinase |
| Abbreviated Name | n/a |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Pyrococcus horikoshii |
| UniProt Accession | O57839_PYRHO |
| 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 | 283 |
| Molecular Weight | 31445.1 |
| Pi | 4.79 |
| Molecular Weight | 31445.1 |
| Disulphides | Unknown |
| Full Sequence |
MLLYTYRSFDMELPTVDIKDADYIILGLPFDGTTSYKPGARFGPVLIRQATLNLESYILDYDIDIAELKIADAGDVALPVSIEDAIKVAVETIKEVRSINPRALPIFLGGEHSMTYPPVKVLEPKSYVVFDAHLDLRDSYQGSRFNHACVARRIHEMGVKVAIFGVRSGTREEVMFASQSGIEWVHARDYNFDAFVDLVSSLPEPVYVSIDVDVFDLPLVPETGTPEPGGLGFWEVIEALEWLTKRKKVAGFDIMEVSGDRLGNSTSITAAKLLFYVIGMSAR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Goda S, Sakuraba H, Kawarabayasi Y, Ohshima T. (2005) Biochemica et Biophysica Acta, 1748, 110-115 |
| Project Aim | Cloning and expression |
| 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 h |
| Expression Vector | pET15b |
| Expression Protocol | Cloning and expression of the gene encoding agmatinase The hybrid plasmid DNA (pUAGM) containing PH0083 (positions 67690–69901 in the entire genome of P. horikoshii) was prepared by the insertion of the gene into the HincII site of pUC118 as previously described [10]. The following set of oligonucleotide primers was used to amplify the agmatinase gene fragment by PCR: the first primer (5′-TGGTGGTCATATGCTACTTTACACGTATCGCTCATT-3′) introduced a unique NdeI restriction site overlapping the 5′ initiation codon, and the other (5′-TCAGGATCCTCACCTAGCACTCATACCTA-3′) introduced a unique BamHI restriction site proximal to the 3′ end of the termination codon. Plasmid pUAGM was used as a template. The amplified 0.9-kb fragment was digested with NdeI and BamHI and then ligated with the expression vector pET15b linearized with NdeI and BamHI to generate pEAGM. The E. coli strain BL21(DE3) codon plus RIL was transformed with pEAGM. The transformants were cultivated at 37 °C in 200 ml of a medium containing 2.4 g of trypton, 4.8 g of yeast extract, 1 ml of glycerol, 2.5 g of K2HPO4, 0.76 g of KH2PO4, and 10 mg of ampicillin, until the optical density at 600 nm reached 0.6. Induction was carried out by the addition of 1.0 mM isopropyl-β-d-thiogalactopyranoside to the medium, and then the cultivation was continued for 3 h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 10 mM potassium phosphate buffer (pH 7.2) containing 10% glycerol, 1 mM EDTA and 0.1 mM DTT |
| Solubilization Buffer | 50 mM Tris/HCl (pH 8.0) buffer with 6 M GuHCl, 200 mM NaCl, and 1 mM EDTA. |
| Refolding Buffer | 0.1 M Tris/HCl (pH 8.0), 0.4 M arginine, 2 mM EDTA and 0.1 mM PMSF |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 40 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Refolding and purification of recombinant agmatinase The E. coli transformed cells were collected (1.65 g wet weight), suspended in 10 mM potassium phosphate buffer (pH 7.2) containing 10% glycerol, 1 mM EDTA and 0.1 mM DTT (buffer A) and disrupted by sonication. After sonication, the suspension was centrifuged at 15,000×g for 20 min at 4 °C. The pellet was solubilized in the 50 mM Tris/HCl (pH 8.0) buffer with 6 M GuHCl, 200 mM NaCl, and 1 mM EDTA. The solubilized agmatinase was added to the refolding buffer (0.1 M Tris/HCl (pH 8.0), 0.4 M arginine, 2 mM EDTA and 0.1 mM PMSF) at 4 °C and was further incubated for about 40 h. Refolded agmatinase was purified by a gel filtration column of Superdex 200 pg (Amersham Bioscience, USA). |
| Refolding Assay | enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.4 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |