Refolding Record:
| Protein | |
|---|---|
| Protein Name | Erbin also known as Protein LAP2 |
| Abbreviated Name | n/a |
| SCOP Family | PDZ domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q96RT1 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | PDZ domain |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 92 |
| Molecular Weight | 9832.2 |
| Pi | 5.29 |
| Molecular Weight | 9832.2 |
| Disulphides | Unknown |
| Full Sequence |
EIRVRVEKDPELGFSISGGVGGRGNPFRPDDDGIFVTRVQPEGPASKLLQPGDKIIQANGYSFINIEHGQAVSLLKTFQNTVELIIVREV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Zhang H, Yu M, Hu M, Qian L, Chen L, Shi M, Shen B, Guo N. (2008) Preparative biochemistry and biotechnology, 38, 282-93 |
| Project Aim | Over expression & Renaturation |
| Fusion | N-terminal +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 | 6 h |
| Expression Vector | pET28a |
| Expression Protocol | E. coli BL21(DE3) (Novagen) transformed with pET-28a-Erbin PDZ was grown at 37°C in 500 mL Luria-Bertani (LB) medium containing 50 µg kanamycin mL-1. The expression was induced with 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at an A600 of 0.4-0.6. The cells were harvested at 6 h postinduction by centrifugation at 15000 g for 20 min at 4°C and washed twice with PBS. The expression of the recombinant protein was analyzed by SDS-PAGE. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.4-0.6 = 600 |
| Cell Disruption Method | Freeze/Thaw+Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 40 mL 0.5% (v/v) Triton X-100 and 1 M urea |
| Solubilization Buffer | 8 M urea |
| Refolding Buffer | 20 mM TrisCl, 1 mM EDTA, 0.2 mM oxidized glutathione, 2 mM reduced glutathione, pH 7.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2/0.2 mM |
| Refolding Protocol | Isolation of Inclusion Bodies and Refolding The cells resuspended in 20 mL equilibration buffer (50 mM sodium phosphate, 500 mM NaCl, pH 7.8) were frozen in liquid nitrogen and thawed at room temperature three times. After centrifugation, the pellet was incubated in lysis buffer (50 mM Tris/HCl, 1 mM EDTA, 0.25 mg lysozyme mL-1, 5 µg DNaseI mL-1, 10 µg RNaseA mL-1, pH 7.8), followed by sonication on ice (3 rounds of 30 s bursts on ice). The insoluble cell fraction was washed twice with 40 mL 0.5% (v/v) Triton X-100 and 1 M urea and then dissolved into 10 mL 8 M urea. Refolding was initiated by ten fold dilution of the denatured proteins dropwise into an ice cold refolding buffer (20 mM TrisCl, 1 mM EDTA, 0.2 mM oxidized glutathione, 2 mM reduced glutathione, pH 7.5) with moderate stirring, followed by dialysis against 1 L of dialysis buffer (20 mM TrisCl, 50 mM NaCl, 0.05 mM oxidized glutathione, 0.5 mM reduced glutathione, pH 7.5) at 4°C overnigh |
| Refolding Assay | Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |