Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interferon Alpha 2a |
| Abbreviated Name | INF-alpha 2a |
| SCOP Family | Interferons/interleukin-10 (IL-10) |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01563 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 165 |
| Molecular Weight | 19241.1 |
| Pi | 5.99 |
| Molecular Weight | 19241.1 |
| Disulphides | 2 |
| Full Sequence |
CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE
|
| Notes | Not specified which varient of IFN actually used. |
| Expression | |
|---|---|
| Report | Babu, K. R., Swaminathan, S., Marten, S., Khanna, N., Rinas, U. (2000) Appl Microbiol Biotechnol, 53, 655-660 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | TG1(DSM 6056) |
| Expression Temp | 42.0 |
| Expression Time | not stated |
| Expression Vector | pMYW |
| Expression Protocol | High density cultivation was carried out at 30degC in a 5-1 bioreactor. Initial batch culture condit |
| Method of Induction | Temperature Shift |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 10mM TrisHCl, pH 8.0, I mM EDTA and I M NaCl |
| Solubilization Buffer | 100mM TrisHCl, pH 8.0, 0.2 mM EDTA, 8M GuHCl |
| Refolding Buffer | 100mM TrisHCl, pH 8.0, 0.5 M L-arginine, 0.2 mM EDTA |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 10.0 |
| Protein Concentration | n/a |
| Refolding Time | 24-36h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Resulting pellet containing Inclusion bodies was then suspended in 10 ml of washing buffer. This was then diluted to 400 ml with more wash buffer and shaken vigorously for 15 min before centrifugation. Inclusion bodies were then washed x2 with distilled H20 and centrifuged. Pellet was then solubilized in 20 ml solubilization buffer and stirred on magnetic stirrer for two hours at room temperature. It then underwent centriguation (17 000rpm, 1hr, 4degC). Supernatant collected and stored at -70degC or refolded. Refolding occured by routine addition 10 ml soluble sollution to 1L of prechilled (10degC) refolding buffer. Three additions were made per leader at 2 h intervals. Refolded material then dialyzed against 50 mM TrisHCl, pH 8.0 containg 1 mM EDA and 100 mM urea for 48 hr at 4 degC with buffer change every 12 h to remove arginine. |
| Refolding Assay | 8-Anilo-1-Naphthalenesulfonic Acid Fluorescence |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.5 M |
| Refolding Yield | 300mg protein/L |
| Purity | n/a |
| Notes | n/a |