Refolding Record:
| Protein | |
|---|---|
| Protein Name | Alfa- glucosidase |
| Abbreviated Name | AGLU |
| SCOP Family | alpha-Amylases, C-terminal beta-sheet domain |
| Structure Notes | |
| Organism | S. cerevisiae |
| UniProt Accession | P53341 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 584 |
| Molecular Weight | 68094.3 |
| Pi | 5.47 |
| Molecular Weight | 68094.3 |
| Disulphides | Unknown |
| Full Sequence |
MTISDHPETEPKWWKEATIYQIYPASFKDSNNDGWGDLKGITSKLQYIKDLGVDAIWVCPFYDSPQQDMGYDISNYEKVWPTYGTNEDCFELIDKTHKLGMKFITDLVINHCSTEHEWFKESRSSKTNPKRDWFFWRPPKGYDAEGKPIPPNNWKSFFGGSAWTFDETTNEFYLRLFASRQVDLNWENEDCRRAIFESAVGFWLDHGVDGFRIDTAGLYSKRPGLPDSPIFDKTSKLQHPNWGSHNGPRIHEYHQELHRFMKNRVKDGREIMTVGEVAHGSDNALYTSAARYEVSEVFSFTHVEVGTSPFFRYNIVPFTLKQWKEAIASNFLFINGTDSWATTYIENHDQARSITRFADDSPKYRKISGKLLTLLECSLTGTLYVYQGQEIGQINFKEWPIEKYEDVDVKNNYEIIKKSFGKNSKEMKDFFKGIALLSRDHSRTPMPWTKDKPNAGFTGPDVKPWFLLNESFEQGINVEQESRDDDSVLNFWKRALQARKKYKELMIYGYDFQFIDLDSDQIFSFTKEYEDKTLFAALNFSGEEIEFSLPREGASLSFILGNYDDTDVSSRVLKPWEGRIYLVK
|
| 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 | None |
| Protein Expression and Production | Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host | None |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 0 |
| Expression Vector | |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | N/A |
| Solubilization Buffer | 6 M guanidine hydrochloride for 1 h at 25˚C |
| Refolding Buffer | 0.5 μM GroEL, 1 μM GroES, 2 mM nucleotide, 10 mM magnesium acetate and 5 mM β-mercaptoethanol. |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 25.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 5 mM |
| Refolding Protocol | AGLU refolding assay AGLU [EC 3.2.1.20] from S. cerevisiae (Sigma), which is too large to be encapsulated in the GroEL/GroES cavity, is a monomeric protein with a molecular mass of 68,000. It hydrolyzes an α-glucosyl bond present in its substrates. AGLU (10 μM) was denatured with 6 M guanidine hydrochloride for 1 h at 25˚C. The unfolded AGLU was diluted 100-fold at 25˚C into the refolding buffer containing 0.5 μM GroEL, 1 μM GroES, 2 mM nucleotide, 10 mM magnesium acetate and 5 mM β-mercaptoethanol. AGLU activity was assayed at 25˚C by monitoring absorbance at 405 nm, resulting from the release of p-nitrophenol in the AGLU assay solution (100 mM sodium phosphate buffer, pH 6.8 and 2 mM p-nitrophenyl-α-D-glucopyranoside). GroEL/EC GroES completely arrested the refolding reaction. Similar events were also observed when either CP GroEL1, or EC GroEL alone was added to the refolding solution in the absence of ATP (Fig. 1d). |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | GroEL,GroES |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | Note in this paper refolding pH isn't specified |