Refolding Record:
| Protein | |
|---|---|
| Protein Name | Adenylate kinase |
| Abbreviated Name | ADK |
| SCOP Family | Nucleotide and nucleoside kinases |
| Structure Notes | |
| Organism | Pig (Sus scrofa) |
| UniProt Accession | P00571 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 195 |
| Molecular Weight | 21638.9 |
| Pi | 8.38 |
| Molecular Weight | 21638.9 |
| Disulphides | Unknown |
| Full Sequence |
MEEKLKKSKIIFVVGGPGSGKGTQCEKIVQKYGYTHLSTGDLLRAEVSSGSARGKMLSEIMEKGQLVPLETVLDMLRDAMVAKVDTSKGFLIDGYPREVKQGEEFERKIGQPTLLLYVDAGPETMTKRLLKRGETSGRVDDNEETIKKRLETYYKATEPVIAFYEKRGIVRKVNAEGSVDDVFSQVCTHLDTLK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hibino T, Misawa S, Wakiyama M, Maeda S, Yazaki K, Kumagai I, Ooi T, Miura K. (1994) J Biotechnology, 32, 139-148 |
| Project Aim | Structural Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JM109 |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | pKAK1-2 |
| Expression Protocol | The overnight culture of E. coli JM109 harboring pMKAK1-2 was harvested by centrifugation, washed with 0.5% NaCI, 0.5% KCI solution and recovered. Then the cells were suspended in 2 vol. of 20 mM Tris-HC1 at pH 7.4, 100 mM NaC1, 1 mM /3-mercaptoethanol, 0.1 mM EDTA and disrupted by sonication. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 0.5% NaCI, 0.5% KCI solution |
| Solubilization Buffer | 6 M guanidine-hydrochloride, 20 mM Tris, pH 7.4, 0.1 mM EDTA |
| Refolding Buffer | Tris-HC1 buffer, pH 7.4, 0.1 mM EDTA |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.4 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The insoluble materials were recovered from the cell lysate by centrifugation at 8000 × g for 20 min. Washed with 5% Triton X-50 solution, the inclusion bodies were recovered by centrifugation at 8000 × g for 10 min. Then the included ADK was solubilized in 6 M guanidine-hydrochloride, 20 mM Tris at pH 7.4, 0.1 mM EDTA. The solution containing ADK was dialyzed against 4 vol. of Tris buffer overnight. The buffer were changed three times after every 4 ~ 6 h during dialysis. After dialysis, the solution containing ADK was charged on Affigel Blue colummn (15 x 1.2 cm) pre-equilibrated with 20 mM MOPS at pH 7.2, 100 mM NaCI, 1 mM DT-I\', 0.1 mM EDTA. Then the column was washed with 5 column volume of MOPS buffer, and ADK was eluted out with MOPS buffer by applying the linear gradient of NaC1 concentration of 0.1 M to 3 M. Every fraction of Affigel chromatography was monitored by 280 nm ultraviolet absorption and several of them were assayed with PK/LDH coupled ADK assay system (Agarwal et al., 1979) at the substrate concentration of 100/xM of both ATP and AMP. The fractions containing ADK were combined and concentrated by ultrafiltration on Amicon concentration apparatus. The concentrated ADK solution was charged on Sephacryl S-200 column (1.2 x 120 cm) pre-equilibrated with Tris buffer and eluted out with the same buffer, all the fractions were monitored by its 280 nm ultraviolet absorption and several fractions were also assayed with the ADK activity at the same conditions as above. The fractions containing ADK were combined and used for further characterization. The purity of the isolated ADK was checked by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970). |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |