Refolding Record:
| Protein | |
|---|---|
| Protein Name | Glucose 6-phosphate dehydrogenase |
| Abbreviated Name | G6PD |
| SCOP Family | Glyceraldehyde-3-phosphate dehydrogenase-like, N-terminal domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P11413 |
| 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 | 515 |
| Molecular Weight | 59125.6 |
| Pi | 6.4 |
| Molecular Weight | 59125.6 |
| Disulphides | Unknown |
| Full Sequence |
AEQVALSRTQVCGILREELFQGDAFHQSDTHIFIIMGASGDLAKKKIYPTIWWLFRDGLLPENTFIVGYARSRLTVADIRKQSEPFFKATPEEKLKLEDFFARNSYVAGQYDDAASYQRLNSHMNALHLGSQANRLFYLALPPTVYEAVTKNIHESCMSQIGWNRIIVEKPFGRDLQSSDRLSNHISSLFREDQIYRIDHYLGKEMVQNLMVLRFANRIFGPIWNRDNIACVILTFKEPFGTEGRGGYFDEFGIIRDVMQNHLLQMLCLVAMEKPASTNSDDVRDEKVKVLKCISEVQA
NNVVLGQYVGNPDGEGEATKGYLDDPTVPRGSTTATFAAVVLYVENERWDGVPFILRCGKALNERKAEVRLQFHDVAGDIFHQQCKRNELVIRVQPNEAVYTKMMTKKPGMFFNPEESELDLTYGNRYKNVKLPDAYERLILDVFCGSQMHFVRSDELREAWRIFTPLLHQIELEKPKPIPYIYGSRGPTEADELMKRVGFQYEGTYKWVNPHKL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Huang Y, Choi MY, Au SW, Au DM, Lam VM, Engel PC. (2007) Molecular genetics and metabolism, 93, 44-53 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DF213 |
| Expression Temp | 25.0 |
| Expression Time | 48 h |
| Expression Vector | pTrc99A |
| Expression Protocol | Transformed cells were grown in Terrific Broth (TB) or Luria-Bertani medium (LB) with ampicillin at different temperatures. For 37 °C expression, cells were grown for 24 h after induction with isopropyl-1-thio-B-d-galactopyranoside (IPTG); for 31 °C expression, cells were grown for 48 h after induction. For expression with pET as expression vector, cells were grown at 37, 30 or 25 °C until OD600nm reached 2.1–2.3. For co-expression with chaperonins GroEL and GroES, plasmid pGroESL was transformed into competent cells of E. coli DF213 harbouring the appropriate G6PD expression plasmids, e.g. pTrc99A/G6PD, pTrc99A/G6PDMahidol or pTrc99A/G6PDPlymouth. The E. coli DF213 cells harbouring the two expression plasmids were grown in TB as previously described in the presence of both ampicillin and chloramphenicol. After induction and further growth, cells were centrifuged at 4000 rpm and 4 °C for 30 min. The pelleted cells were resuspended in extraction buffer A (0.1 M Tris–Cl, pH 7.6, 3 mM MgCl2, 5 mM EDTA, 0.5 mM PMSF, 1 mM aminocaproic acid, 3 μg/ml aprotinin, 0.1% B-mercaptoethanol, 1 μM NADP+) and lysed by sonication. The crude extract was centrifuged at 16,000 rpm and 4 °C for 2 h and the supernatant was used for purification of the enzyme. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 2.1-2.3 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 1 M Tris–Cl, pH 7.6, 3 mM MgCl2, 5 mM EDTA, 0.5 mM PMSF, 1 mM aminocaproic acid, 3 ug/ml aprotinin, 0.1% B-mercaptoethanol, 1 uM NADP+ |
| Solubilization Buffer | 10 mM Tris–Cl, pH 7.4, 2 mM EDTA |
| Refolding Buffer | 10 mM potassium phosphate, pH 7.4 (buffer P) |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.4 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 0.1% |
| Refolding Protocol | Unfolding and refolding study Unfolding was carried out with 12 ug/ml enzyme, 0–5.0 M urea and 0–1000 uM NADP+ in buffer T. The mixture was incubated at 25 °C for 1 h. Residual activity was determined and expressed as a percentage of the enzyme activity without urea and NADP+. For refolding experiments, mixtures of 0.6 mg/ml enzyme and 5 M urea were kept at 25 °C for 2 h to fully inactivate the enzyme. The unfolded enzyme was then diluted with 50× volumes of 10 mM potassium phosphate, pH 7.4 (buffer P) or buffer T at 25 °C with or without various additives. One of the additives, GroEL, was from the crude extract of G6PD deficient E. coli DF213 cells expressing GroEL and GroES. Enzyme activity was assayed periodically and the maximum regain of activity is reported. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |