Refolding Record:
| Protein | |
|---|---|
| Protein Name | Der f 1 |
| Abbreviated Name | Der f 1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Dermatophagoides farinae (dust mite) |
| UniProt Accession | P16311 |
| 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 | 304 |
| Molecular Weight | 34644.7 |
| Pi | 5.53588 |
| Molecular Weight | 34644.7 |
| Disulphides | 3 |
| Full Sequence |
MRP ASIKTFEEFK KAFNKNYATV EEEEVARKNF LESLKYVEAN KGAINHLSDL SLDEFKNRYL MSAEAFEQLK TQFDLNAETS ACRINSVNVP SELDLRSLRT VTPIRMQGGC GSCWAFSGVA ATESAYLAYR NTSLDLSEQE LVDCASQHGC HGDTIPRGIE YIQQNGVVEE RSYPYVAREQ RCRRPNSQHY GISNYCQIYP PDVKQIREAL TQTHTAIAVI IGIKDLRAFQ HYDGRTIIQH DNGYQPNYHA VNIVGYGSTQ GDDYWIVRNS WDTTWGDSGY
GYFQAGNNLM MIEQYPYVVI M
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Takahashi K, Takai T, Yasuhara T, Yuuki T, Ohtake Y, Yokota T, Okumura Y (2000) Int Arch Allergy Immunol, 122, 108-114 |
| Project Aim | Drug Studies |
| Fusion | pro sequence |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 30.0 |
| Expression Time | 3-6h |
| Expression Vector | pGEMEX |
| Expression Protocol | Cells were incubated at 30degC until OD600 reached 0.4-0.5. 0.1mM IPTG was added then cells were cultured for a further 3-6h. After centrifugation, the cell pellet was washed with wash buffer and frozen at -80degC. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.4-0.5 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | 100mM TrisCl pH 8.5, 10mM EDTA, 1mM PMSF |
| Solubilization Buffer | 100mM TrisCl pH 8.5, 10mM EDTA, 1mM PMSF, 8M urea, 1% DTT |
| Refolding Buffer | 20mM TrisCl pH 8.6 |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.6 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were disrupted by sonication and centrifuged (15000g, 15min). The pellet was washed with wash buffer. Inclusion bodies were resuspended in solubilization buffer and stirred gently at room temperature for 1-2h. The solubilized protein was then applied to a a Sephacryl S-200 16/60 gel filtration column equilibrated with refolding buffer. The protein was then dialyzed aginst PBS(per L: 8g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4.12H2O, pH 7.4) and then subjected to a second gel filtration step using the same column equilibrated with PBS. Selected fraction(s) were then dialyzed with 100mM acetate buffer (pH 4.0) at 4degC overnight to remove the pro-sequence |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | |