Refolding Record:
| Protein | |
|---|---|
| Protein Name | 60 kDa chaperonin groEL protein |
| Abbreviated Name | groEL |
| SCOP Family | GroEL chaperone, ATPase domain |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | P0A6F5 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Heptamer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 547 |
| Molecular Weight | 57197.7 |
| Pi | 4.84 |
| Molecular Weight | 57197.7 |
| Disulphides | Unknown |
| Full Sequence |
AAKDVKFGNDARVKMLRGVNVLADAVKVTLGPKGRNVVLDKSFGAPTITKDGVSVAREIELEDKFENMGAQMVKEVASKANDAAGDGTTTATVLAQAIITEGLKAVAAGMNPMDLKRGIDKAVTAAVEELKALSVPCSDSKAIAQVGTISANSDETVGKLIAEAMDKVGKEGVITVEDGTGLQDELDVVEGMQFDRGYLSPYFINKPETGAVELESPFILLADKKISNIREMLPVLEAVAKAGKPLLIIAEDVEGEALATLVVNTMRGIVKVAAVKAPGFGDRRKAMLQDIATLTGGTVISEEIGMELEKATLEDLGQAKRVVINKDTTTIIDGVGEEAAIQGRVAQIRQQIEEATSDYDREKLQERVAKLAGGVAVIKVGAATEVEMKEKKARVEDALHATRAAVEEGVVAGGGVALIRVASKLADLRGQNEDQNVGIKVALRAMEAPLRQIVLNCGEEPSVVANTVKGGDGNYGYNAATEEYGNMIDMGILDPTKVTRSALQYAASVAGLMITTECMVTDLPKNDAADLGAAGGMGGMGGMGGMM
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Okuda H, Sakuhana C, Yamamoto R, Kawai R, Mizukami Y, Matsuda K. (2007) Biometals, 20, 903-910 |
| Project Aim | Folding |
| 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 | 12 h |
| Expression Vector | pET22b |
| Expression Protocol | Recombinant GroEL was expressed in E. coli strain BL21 (DE3) (Novagen). The plasmid construct was used to transform E. coli BL21 (DE3), and the transformants were grown at 37°C in LB broth containing ampicillin (100 ug/ml). The protein expression was then induced by the addition of 0.5 mM IPTG. After incubation at 25°C for 12 h, the protein-overexpressing bacterial cells were harvested and resuspended in buffer A (50 mM Tris–HCl, pH 7.5, containing 1 mM EDTA and 50 mM NaCl). Lysozyme (0.5 mg/ml, Nacalai Tesque) and benzonase nuclease (5 units/ml, Novagen) were added to the cell suspension, which was subsequently disrupted by ultrasonication (Sonifire 450, Branson, CT, USA). The supernatant was fractionated by ammonium sulfate precipitation by a stepwise gradient from 30% (w/v) to 65% (w/v) saturation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | ultrasonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | gel filtration |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50 mM Tris–HCl, pH 7.5, containing 1 mM EDTA and 50 mM NaCl |
| Solubilization Buffer | 50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 250 mM NaCl, 20% (v/v) ethanol |
| Refolding Buffer | 50 mM MOPS-KOH, pH 7.0, 10 mM KCl |
| Pre-Refolding Purification | gel filtration |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | The protein precipitate obtained at 65% ammonium sulfate saturation was collected and dissolved in buffer A. The protein solution was dialyzed overnight with 100-fold volume of buffer A with two buffer changes. Dialyzed protein was loaded onto a HiPrep Q anion exchange column (Amersham Biosciences, NJ, USA) with buffer A and eluted by a linear gradient of NaCl from 50 mM to 1 M. Fractions containing GroEL, as determined by SDS-PAGE analysis, were pooled and the protein was concentrated by ultrafiltration using a 100-kDa cutoff filter (Millipore, MA, USA). The concentrated GroEL was purified by gel filtration using a TSK-GEL G4000SW column (column size: 21.5 × 600 mm, TOSOH, Tokyo, Japan) with buffer B (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 250 mM NaCl, 20% (v/v) ethanol). Fractions containing 14 meric GroEL were pooled and concentrated using a 100-kDa cutoff filter. The concentrated GroEL was loaded onto a Mono Q anion exchange column (Amersham Biosciences) with buffer A and eluted with a linear gradient of NaCl from 145 mM to 1 M. The GroEL-containing fractions were pooled and dialyzed with refolding buffer (50 mM MOPS-KOH, pH 7.0, 10 mM KCl). The concentrations of purified GroEL were determined by the Bradford method using bovine serum albumin as the standard, and their purity was checked by SDS-PAGE and native PAGE analysis using Coomassie brilliant blue R-250 (Nacalai Tesque) for protein staining. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None,GroEL |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |