Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interferon regulatory factor 8 |
| Abbreviated Name | IFN-con |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q02556 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 426 |
| Molecular Weight | 48356.1 |
| Pi | 6.37 |
| Molecular Weight | 48356.1 |
| Disulphides | Unknown |
| Full Sequence |
MCDRNGGRRLRQWLIEQIDSSMYPGLIWENEEKSMFRIPWKHAGKQDYNQEVDASIFKAWAVFKGKFKEGDKAEPATWKTRLRCALNKSPDFEEVTDRSQLDISEPYKVYRIVPEEEQKCKLGVATAGCVNEVTEMECGRSEIDELIKEPSVDDYMGMIKRSPSPPEACRSQLLPDWWAQQPSTGVPLVTGYTTYDAHHSAFSQMVISFYYGGKLVGQATTTCPEGCRLSLSQPGLPGTKLYGPEGLELVRFPPADAIPSERQRQVTRKLFGHLERGVLLHSSRQGVFVKRLCQGRVFCSGNAVVCKGRPNKLERDEVVQVFDTSQFFRELQQFYNSQGRLPDGRVVLCFGEEFPDMAPLRSKLILVQIEQLYVRQLAEEAGKSCGAGSVMQAPEEPPPDQVFRMFPDICASHQRSFFRENQQITV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Liu YD, Li JJ, Wang FW, Chen J, Li P, Su ZG. (2006) Protein Expression and Purification, 51, 235-242 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | TOP10 |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pBR322 |
| Expression Protocol | Expression, isolation and purification of inclusion bodies of rIFN-con Bacterial cells carrying rIFN-con1 were first grown at 37 °C and 250 rpm in 1 l shake-flasks containing 500 ml LB medium supplemented with 50 μg/ml kanamycin and then inoculated in a 7 l bioreactor (K&T, Korea) when cell density reached to OD600 of 0.6–0.8. Cells were continuously grown in 5 l LB medium supplemented with 100 μg/ml kanamycin. Protein expression was induced when cell density reached to OD600 of 1.0 with 1 mM isopropyl-d-thiogalactopyranoside (IPTG). After 4 h induction, cells were harvested by centrifugation at 4,000 rpm for 30 min at 4 °C. One hundred and ten grams wet weight cells were recovered and frozen at −20 °C. Ten grams collected wet cells was taken and suspended in 100 ml of 25 mM Tris–HCl, pH 8.0, containing 1 mM EDTA. Cell suspensions were sonicated at 150 kHz, using VC-600-2 sonicator (Sonics & Materials INC) with a 13 mm probe. This cycle was repeated 6× for a total sonication time of 6 min. Cell debris were removed by centrifugation at 12,000 rpm for 20 min at 4 °C. The pellets were suspended with 50 ml of 25 mM Tris–HCl, pH 8.0, containing 1 mM EDTA, 2 M urea, 0.5% Triton X-100 and then centrifuged again. The pellets were resuspended and washed several times with 50 ml of the same buffer. One gram wet weight of inclusion bodies were obtained with a purity of more than 95% upon SDS–PAGE analysis with Coomassie blue staining. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 1 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 ml of 25 mM Tris–HCl, pH 8.0, containing 1 mM EDTA, 2 M urea, 0.5% Triton X-100 |
| Solubilization Buffer | 10 ml of 100 mM Tris–HCl, pH 8.5, containing 6 M Guanidine chloride, 50 mM 2-mercaptoethanol, and 1 mM EDTA |
| Refolding Buffer | 100 mM Tris–HCl, pH 8.5, containing 0.1 mM EDTA, and various concentrations of arginine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 30 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Refolding of rIFN-con1 with different concentration of arginine Refolding was initiated by rapid 100-fold dilution of the denatured protein into refolding buffer of 100 mM Tris–HCl, pH 8.5, containing 0.1 mM EDTA, and various concentrations of arginine. Aggregation during refolding was measured on the turbidity represented by absorbance at 340 nm. A fast and efficient mix is essential for the first few minutes and then the solution was incubated for 30 h at room temperature without any agitation. The refolding solution was centrifuged at 12,000 rpm for 20 min at 4 °C and the supernatant was subjected to the following analysis. |
| Refolding Assay | SDS-PAGE,RP-HPLC,Oligomeric protein analysis |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | Various concentrations |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |