Refolding Record:
| Protein | |
|---|---|
| Protein Name | 10 kDa chaperonin groES protein |
| Abbreviated Name | groES |
| SCOP Family | GroES |
| Structure Notes | |
| Organism | Chlamydolphila pneumoniae |
| UniProt Accession | P31682 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Heptamer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 102 |
| Molecular Weight | 11316.0 |
| Pi | 4.64 |
| Molecular Weight | 11316.0 |
| Disulphides | Unknown |
| Full Sequence |
MSDQATTLRIKPLGDRILVKREEEEATARGGIILPDTAKKKQDRAEVLVLGTGKRTDDGTLLPFEVQVGDIILMDKYAGQEITIDDEEYVILQSSEIMAVLK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Okuda H, Sakuhana C, Yamamoto R, Mizukami Y, Kawai R, Sumita Y, Koga M, Shirai M, Matsuda K. (2008) Biologycal Chemistry, 1, 1 |
| Project Aim | Folding |
| Fusion | C-terminal hexahistidine tag |
| 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 |
| Expression Vector | pET22b |
| Expression Protocol | goEL1 and groES genes from C.pneumoniae strain J138 were cloned into the pET22b (+) vector (Novagen). The metf gene fused with His-tag sequences at the 3’-end was also cloned into the pET22b (+) vector. Moreover, a series of the groEL chimera genes with exchanged apical, intermediate and equatorial domains of the groEL gene from E. coli and the groEL1 gene from C. pneumoniae were constructed by an overlap extension method using PCR (18) and subcloned into the pET22b (+) vector. Mutant groEL genes were also constructed by DpnI mediated site-directed mutagenesis and subcloned into the pET22b (+) vector. All genes were ligated into NdeI and XhoI sites of the pET22b (+) vector. The primer sets for gene cloning, chimera construction and mutagenesis using PCR are listed in Tables S1, S2 and S3, respectively, in Supplemental Data. Moreover, schematic representations of primer annealing sites for the preparation of the chimeric genes are shown in Fig. S4 in Supplemental Data. Protein Purification The recombinant proteins, GroELs, GroESs and METF, were purified as described previously (19). Molecular masses of EC GroEL, CP GroEL1 and their chimeras were confirmed by MALDI-TOF mass spectrometry (MALDI micro MX, Waters Japan, Tokyo, Japan). |
| 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 |
| 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 precipitates resulting from 55% ammonium sulfate saturation were harvested and dissolved in buffer A. The protein solution was then dialyzed overnight with 100-fold volume of buffer A with two buffer changes. The dialyzed proteins were loaded onto a HiPrep Q anion exchange column with buffer A and eluted by a linear gradient of NaCl from 50 mM to 1 M. Fractions containing GroES were pooled and concentrated by ultrafiltration using a 10-kDa cutoff filter (Millipore). Concentrated GroES was dialyzed overnight with 100-fold volumes of buffer C (50 mM acetic acid buffer, pH 5.3) with two buffer changes and the insoluble matter was removed from the dialyzed proteins by centrifugation. The resultant supernatants were loaded onto a Mono Q anion exchange column with buffer C and eluted with a linear gradient of NaCl from 0 mM to 400 mM. Moreover, the fraction containing GroES were purified using a Mono Q anion exchange column with 4-column volume of buffer A. The proteins were eluted with a linear gradient of NaCl from 50 mM to 1 M. Finally, GroES-containing fractions were purified by gel filtration using a Superdex 200 GL column (Amersham Biosciences) with buffer B. The fractions containing 7 meric GroES proteins were pooled and dialyzed with refolding buffer. The purity of the proteins was checked by SDS and native PAGE as described for GroEL. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | GroES |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | For Refolding protocol please see Okuda H et al (2007) Biometals |