Refolding Record:
| Protein | |
|---|---|
| Protein Name | Fusion protein consisting of interleukin 13 and pseudomonas exotoxin |
| Abbreviated Name | IL-13PE |
| SCOP Family | Short Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P35225-P11439 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 727 |
| Molecular Weight | 79081.4 |
| Pi | 5.6 |
| Molecular Weight | 79081.4 |
| Disulphides | Unknown |
| Full Sequence |
GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFNAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYG
YAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Joshi BH, Puri RK. (2005) Protein Expression and Purification, 39, 189-198 |
| Project Aim | Purification & characterization |
| 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 | 1-8 h |
| Expression Vector | PET24a |
| Expression Protocol | One hundred microliters fresh transformed bacterial preparation was added to 100ml of enriched superbroth (Biowhittakar, Walkersville, MD) that contained 15􏱿g/ml kanamycin, 20ml of 20% glucose, and 12ml of 4% magnesium sulfate. The bacteria were induced with 0.25–2.5mM IPTG when the optical density of the bacteria reached between 0.5 and 0.6 OD and shaken for an additional 1–8h in a bacterial shaker at 37°C. One milliliter of the bacteria was taken out at 1, 2, 3, 4, 5, 6, and 8h, centrifuged, and lysed. Five microliters of cell lysate was electrophoresed on a 10% nonreducing PAGE for evaluation of protein expression. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5-0.6 = n/a |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | three times with 50/20mM Tris buVer with 0.1% Triton X-100 and two additional washings with 50/20mM Tris buVer without Triton X-100 |
| Solubilization Buffer | 10ml of 7M guanidinium–hydrochloride dissolved in 50mM Tris–HCl, pH 8.0, and 65mM dithiothreitol (DTE) |
| Refolding Buffer | 0.6M arginine and 0.9mM oxidized glutathione |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 10.0 |
| Protein Concentration | n/a |
| Refolding Time | 48 h |
| Redox Agent | GSSG |
| Redox Agent Concentration | 0.9 mM |
| Refolding Protocol | We next optimized refolding step of the solubilized inclusion bodies by refolding 5, 10, 15, or 20mg protein/ ml diluted to 33.3􏱿g–133.3􏱿g/ml in refolding buVer. Refolding buVer that contained 0.6M arginine and 0.9mM oxidized glutathione was used for refolding of denatured protein at 10°C for 48h. The refolded preparation was dialyzed against 10mM Tris–HCl, pH 7.4, buVer containing 60mM urea. The chimeric protein was puriWed by Fast Protein Liquid Chromatography using Q-Sepharose, mono Q, and Sephacryl S-100 gel exclusion columns (Amersham–Pharmacia, Piscataway, NJ). The puriWed protein was electrophoresed on a 10% SDS–PAGE gel and stained with Coomassie blue. The gel was destained with 7% acetic acid and 5% methanol (v/v). Both IL-13-PE38QQR and IL-13-PE38 proteins appeared to be induced equally well with IPTG and puriWed to single band entities, demonstrating high purity of the protein products. The chimeric proteins migrated at approximately 50kDa as expected. |
| Refolding Assay | Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.6 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |