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 | 2 |
| Full Sequence |
MCDRNGGRRLRQWLIEQIDSSMYPGLIWENEEKSMFRIPWKHAGKQDYNQEVDASIFKAWAVFKGKFKEGDKAEPATWKTRLRCALNKSPDFEEVTDRSQLDISEPYKVYRIVPEEEQKCKLGVATAGCVNEVTEMECGRSEIDELIKEPSVDDYMGMIKRSPSPPEACRSQLLPDWWAQQPSTGVPLVTGYTTYDAHHSAFSQMVISFYYGGKLVGQATTTCPEGCRLSLSQPGLPGTKLYGPEGLELVRFPPADAIPSERQRQVTRKLFGHLERGVLLHSSRQGVFVKRLCQGRVFCSGNAVVCKGRPNKLERDEVVQVFDTSQFFRELQQFYNSQGRLPDGRVVLCFGEEFPDMAPLRSKLILVQIEQLYVRQLAEEAGKSCGAGSVMQAPEEPPPDQVFRMFPDICASHQRSFFRENQQITV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Liu YD, Zhang GF, Li JJ, Chen J, Wang YJ, Ding H, Su ZG. (2007) Biotechnol Appl Biochem., 48, 189-198 |
| 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 | E. coli strain TOP10 cells transformed with vector pBR322 carrying the rIFN-con1 gene (provided by Institute of Microbiology, Chinese Academy of Sciences) were used to express rIFN-con1 [21].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/Dialysis combination |
| 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 | 100 mM Tris/HCl, pH 8.5, containing 6 M GdmCl, 50 mM 2-ME and 1 mM EDTA |
| Refolding Buffer | 100 mM Tris/HCl, pH 8.5, containing 0.1 mM EDTA and 0.5 M arginine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 5 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Two-stage refolding strategy Inclusion bodies were dissolved in a denaturant buffer containing 700 mM 2-ME. First, the denatured inclusion bodies were refolded using the dilution method as described above and incubated at 4 °C for 5 h. Secondly, the folded sample was loaded on to a dialysis cassette with a membrane molecular mass cutoff of 10000–12000 Da. The cassette was dialysed against 50 mM Tris/HCl (pH 8.5) containing 1 mM EDTA, 0.25 mM arginine and 1 mM GSSG for 12 h at 4 °C, followed by another dialysis against 50 mM Tris/HCl (pH 8.5) containing 1 mM EDTA and 3 mM GSSG for 12 h at 4 °C. The sample was recovered from the dialysis cassette and stored at 4 °C prior to analysis. |
| Refolding Assay | RP-HPLC |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.5 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |