Refolding Record:
| Protein | |
|---|---|
| Protein Name | Der f2 |
| Abbreviated Name | Der f2 |
| SCOP Family | E-set domains of sugar-utilizing enzymes |
| Structure Notes | |
| Organism | Dermatophagoides farinae (dust mite) |
| UniProt Accession | Q00855 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 129 |
| Molecular Weight | 14076.2 |
| Pi | 6.45841 |
| Molecular Weight | 14076.2 |
| Disulphides | 3 |
| Full Sequence |
DQV DVKDCANNEI KKVMVDGCHG SDPCIIHRGK PFTLEALFDA NQNTKTAKIE IKASLDGLEI DVPGIDTNAC HFMKCPLVKG QQYDIKYTWN VPKIAPKSEN VVVTVKLIGD NGVLACAIAT HGKIRD
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Nishiyama C, Yuuki T, Usui Y, Iwamoto N, Okumura Y, Okudaira H (1994) Int Arch Allergy Immunol, 105, 62-69 |
| Project Aim | Structural 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 | 30.0 |
| Expression Time | 4h |
| Expression Vector | pGEMEX1 |
| Expression Protocol | 170ml of LB media was inoculated with 1.7ml of an overnight LB culture of cells, with 50 micrograms/ml ampicillin. After 2.5h incubation at 30degC, expression was induced with 1mM IPTG. After a further 4h incubation, the cells were harvested by centrifugation and resuspended in 6ml x 100mM TrisHCl, 10mM EDTA, pH 7.5. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M urea, 20mM TrisHCl pH 8.5 |
| Refolding Buffer | 20mM Tris-HCl pH 8.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 12h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were sonicated, then the lysate was centrifuged (15min x 7000g) and the inclusion bodies were solubilized in 50ml solubilization buffer. The protein was then dialyzed againse refolding buffer for 12h. The refolded protein was then applied to a DEAE-Toyopearl column equilibrated with refolding buffer, and eluted with 20mM TrisHCl (pH 8.5) with 50mM NaCl. |
| Refolding Assay | HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 10mg/L |
| Purity | |
| Notes | See also Iwamoto et al (1996) Int Arch Allergy Immunol. 1996 Apr;109(4):356-61. (PubMed: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=8634520) for expression/purification |