Refolding Record:
| Protein | |
|---|---|
| Protein Name | Flotillin-1 |
| Abbreviated Name | FLOT1 |
| SCOP Family | Band 7/SPFH domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | O75955 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 427 |
| Molecular Weight | 47355.3 |
| Pi | 7.07 |
| Molecular Weight | 47355.3 |
| Disulphides | Unknown |
| Full Sequence |
MFFTCGPNEAMVVSGFCRSPPVMVAGGRVFVLPCIQQIQRISLNTLTLNVKSEKVYTRHGVPISVTGIAQVKIQGQNKEMLAAACQMFLGKTEAEIAHIALETLEGHQRAIMAHMTVEEIYKDRQKFSEQVFKVASSDLVNMGISVVSYTLKDIHDDQDYLHSLGKARTAQVQKDARIGEAEAKRDAGIREAKAKQEKVSAQYLSEIEMAKAQRDYELKKAAYDIEVNTRRAQADLAYQLQVAKTKQQIEEQRVQVQVVERAQQVAVQEQEIARREKELEARVRKPAEAERYKLERLAEAEKSQLIMQAEAEAASVRMRGEAEAFAIGARARAEAEQMAKKAEAFQLYQEAAQLDMLLEKLPQVAEEISGPLTSANKITLVSSGSGTMGAAKVTGEVLDILTRLPESVERLTGVSISQVNHKPLRTA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Ding Y, Jiang M, Jiang W, Su Y, Zhou H, Hu X, Zhang Z. (2005) Protein Expression and Purification, 42, 137-145 |
| Project Aim | Identification and Characterization |
| Fusion | N-terminal +C terminal hexahistidine tag + thioredoxin tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 (DE3) or HMS174(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 5 h |
| Expression Vector | pET32a |
| Expression Protocol | Expression four types of flotillin-1 fusion proteins The expression vectors of pT7470-Flot1, pET32a-Flot1, pGEX-Flot1, and pET43.1a-Flot1 were transformed into BL21 (DE3) or HMS174 (DE3) for expression. After grown overnight at 37 °C in 20 ml LB medium supplemented with 100 μg/ml ampicillin, a portion (2 ml) of the bacterial suspension was then transferred into 1 L fresh LB medium and grew in 37 °C shaker (240 rpm) to OD (600 nm) of 0.6, then the cells were cooled down to 25 °C. Expression of the flotillin-1 fusion proteins was induced for 5 h by adding IPTG to a final concentration of 0.1 mM. The cells were collected by centrifugation at 6000g for 15 min and were frozen at −20 °C until use. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | 50 mM Tris–HCl, pH 8.0, 1% Triton X-100, and 200 μg/ml PMSF |
| Solubilization Buffer | 8 M Urea, 50 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM BME, and 200 μg/ml PMSF) containing 10 mM imidazole |
| Refolding Buffer | 50 mM Tris–HCl, pH 8.0, 100 mM NaCl, 0.1% OG, 1% Tween 20, 1 mM BME, and 200 μg/ml PMSF) containing 10 mM imidazole |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cells were thawed and resuspended in 200 ml buffer A (50 mM Tris–HCl, pH 8.0, 100 mM NaCl, and 200 μg/ml PMSF) and were lysed by sonication. The cellular debris was collected by centrifugation at 20,000g for 20 min and washed by buffer B (50 mM Tris–HCl, pH 8.0, 1% Triton X-100, and 200 μg/ml PMSF) for 3 times. Finally, the washed inclusion bodies were solubilized in 40 ml buffer C (8 M Urea, 50 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM BME, and 200 μg/ml PMSF) containing 10 mM imidazole with gentle circular agitation for 2 h at 4 °C. After centrifugation at 20,000g for 20 min, the supernatant fraction was collected and loaded on a 10 ml (15 × 1 cm) Ni–NTA Superflow column already equilibrated with buffer C containing 10 mM imidazole. Then the column was washed by 20 ml buffer C containing 20 mM imidazole and was ready for the refolding on column. The linear gradient formed with buffer C containing 10 mM imidazole (100–0%) and buffer D (50 mM Tris–HCl, pH 8.0, 100 mM NaCl, 0.1% OG, 1% Tween 20, 1 mM BME, and 200 μg/ml PMSF) containing 10 mM imidazole (0–100%), the flow rate was 0.5 ml/min and total volume was 100 ml. The refolded TRX-Flot1 protein was eluted with buffer D containing 300 mM imidazole and loaded on a Sephadex G-50 column equilibrated with buffer D (without PMSF) to remove imidazole and PMSF. Thioredoxin (TRX) tag was digested by thrombin (5 U/ml) at 8 °C for 12 h. The TRX tag and uncleaved TRX-Flot1 were removed by passing through Ni–NTA Superflow column again. The sample was loaded on a 5 ml High Q anion exchange column equilibrated with buffer D and eluted by the linear gradient formed by buffer D and buffer D containing 300 mM NaCl in flow rate of 0.5 ml/min and total volume of 20 ml. The flotillin-1 peak was collected and concentrated using Amicon Ultra-15 centrifugal filter before loading on Sephacryl 200 column (60 × 1 cm) equilibrated with buffer E (50 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1% OG, 1 mM DTT, 1 mM EDTA, and 200 μg/ml PMSF). The peak was collected, concentrated, and stored at 4 °C. |
| Refolding Assay | Western Blot,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | tween 20 |
| Additives Concentration | 1% |
| Refolding Yield | 3.4 mg |
| Purity | 98% |
| Notes | n/a |