Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interleukin-13 |
| Abbreviated Name | IL-13 |
| SCOP Family | Short Chain Cytokines |
| Structure Notes | |
| Organism | Dog (Canis familiaris) |
| UniProt Accession | Q9N0W9 |
| 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 | 131 |
| Molecular Weight | 14267.7 |
| Pi | 8.95 |
| Molecular Weight | 14267.7 |
| Disulphides | 0 |
| Full Sequence |
MALWLTVVIA LTCLGGLASP SPVTPSPTLK ELIEELVNIT QNQASLCNGS MVWSVNLTAG MYCAALESLI NVSDCSAIQR TQRMLKALCS QKPAAGQISS ERSRDTKIEV IQLVKNLLTY VRGVYRHGNF R
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Yang S, Boroughs KL, McDermott (2000) J Interferon and Cytokine Research, 20, 779-785 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 42.0 |
| Expression Time | 5h |
| Expression Vector | p-lambda-cro |
| Expression Protocol | Cells were grwon in LB broth containing 100microg/ml ampicillin. 1L cultures were inoculated with 10ml overnight cultures and grown at 30degC until OD600 reached 0.8-1. Expression was induced by shifting the temperature to 42degC and cultures were incubated for an additional 5h. Cells were chilled then harvested by centrifugation (6000g, 20min) |
| Method of Induction | Temperature Shift |
| Cell Density at Induction | OD 600 = 0.8-1 |
| Cell Disruption Method | Microfluidizer |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 25mM Tris pH 7.5, 1% Triton X-100, 1% deoxycholate |
| Solubilization Buffer | 1: 25mM Tris pH 9.5, 8M urea, 100mM 2-mercaptoethanol, 2: 0.1M Tris pH 8.0, 6M GdnHCl, 2mM EDTA, 0.3M DTT |
| Refolding Buffer | 0.1M Tris pH 8, 0.5M L-arginine, 8mM oxidized glutathione, 2mM EDTA |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.3mg/ml |
| Refolding Time | overnig |
| Redox Agent | GSSG |
| Redox Agent Concentration | 8mM |
| Refolding Protocol | Harvested cells (3g wet weight/L) were suspended in 25ml Tris pH 7.5 using a homogenizer, then passed five times through a microfluidizer at 120psi. The cell lysate was centrifuged (30000g, 30min, 4degC). The pellet was resusupended in 25ml wash buffer, homogenized and recentrifuged (30000g, 30min). The inclusion bodies were then solubilized by homogenization in solubilization buffer 1 and incubated for 60min with gentle mixing at ambient temperature. Insoluble material was removed by centrifugation (30000g, 30min) and the supernatant was adjusted to pH 4.5 with HCl and gently stirred for 90min on ice. The protein precipiated during the step and was recovered by centrifugation. The precipitated protein was resuspended in solubilization buffer 2 with gentle mixing at ambient temperature for 2h. Protein concentration was adjusted to approximately 3mg/ml. The protein was then refolded by rapid dilution with 100-fold volume of refolding buffer. The solution was stirred gently overnight at 4deg, then insoluble material was removed by centrifugation. The supernatant was dialyzed extensively against PBS then concentrated. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.5M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |