Refolding Record:
| Protein | |
|---|---|
| Protein Name | Delta-like1 |
| Abbreviated Name | Dll1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | O00548 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | hDll1 (127–225) |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 98 |
| Molecular Weight | 11229.3 |
| Pi | 5.23 |
| Molecular Weight | 11229.3 |
| Disulphides | Unknown |
| Full Sequence |
HTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPI
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Shi ZX, He F, Wang LL, Liang YM, Han H, Wang CZ, Zhao Q, Geng XD. (2008) Protein Expression and Purification, 1, 1 |
| 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 | DH5a |
| Expression Temp | 34.0 |
| Expression Time | overnight |
| Expression Vector | pGEX-hDll1 |
| Expression Protocol | Escherichia coli DH5α transformed with pGEX4T-1 or pGEX-hDll1 was inoculated in 10 ml of Luria–Bertani (LB) medium supplemented with ampicillin (100 μg/ml), and was cultured at 34 °C with shaking (210 rpm) overnight. On the next day, the bacterial suspension (0.5 ml) was transferred into 50 ml fresh LB medium in a 250 ml shake flask, and was cultured at 30 °C further until OD600 reached 0.8. Expression of recombinant protein was induced by adding 1 M isopropyl-β-d-thiogalactoside (IPTG) in the culture medium to a final concentration of 0.6 mM, and was monitored by SDS–15% PAGE. For large-scale production of recombinant proteins, 400 ml of overnight bacterial culture was inoculated into 4-L of culture media in a CT-5l bioreactor (B. Braun, Germany), followed by fed-batch fermentation at 34 °C. During the fermentation, pH was controlled at 7.0 by addition of 5 M NaOH, and dissolved oxygen, which was monitored using a polarographic electrode (B. Braun), was controlled at 20–30% of air saturation by controlling both airflow and stirring speed. During the fed-batch phase, the inlet air was enriched with pure oxygen. Foam was controlled by the addition of silicon-antifoaming reagent. After 5 h of fermentation, culture was induced by addition of IPTG to a final concentration of 0.6 mM. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | 50 mM phosphate buffer, pH 7.4, 1 mM EDTA, 0.5 mM NaCl, and 2 M urea |
| Solubilization Buffer | 4.5 ml 8 M urea, and finally 9.22 mg/ml |
| Refolding Buffer | 8 M urea (100% mobile phase B (20 mM KH2PO4, pH 7.0, 0.1 M NaCl, 8 M urea) ) to 2 M urea (25% mobile phase B |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | SEC and HP-RPLC was carried out using an LC–20ATvp high-performance liquid chromatography system (Shimadzu, Kyoto, Japan) consisting of two pumps (LC–20A), a variable-wavelength UV–vis detector (SPD–20AV), and a system controller (SCL–10B). The SEC column (Superdex-75 media, 20 × 1.0 cm i.d.) was equilibrated using 100% mobile phase A (20 mM KH2PO4, pH 7.0, 0.1 M NaCl) at each selected flow-rates before injecting sample solutions. The dissolved 100 μl protein sample (45 mg pure inclusion body was dissolved in 4.5 ml 8 M urea, and finally 9.22 mg/ml) was injected into the column through the sample valve, and a linearly decreasing urea gradient from 8 M urea (100% mobile phase B) to 2 M urea (25% mobile phase B) in the mobile phase B (20 mM KH2PO4, pH 7.0, 0.1 M NaCl, 8 M urea) was applied for elution. The urea gradient-eluted proteins were collected in fractions, desalted by reverse phase-high-performance liquid chromatography (RP–HPLC; SP-300-10-C4 from SUNCHROM, 150 × 4.6 mm, i.d.), and lyophilized for further analyses. AFC was carried out using an ÄKTA Explorer 100A chromatographic system with a UV-900 detector. Chromatographic data were evaluated using the Unicorn 3.21 Data system. GSTrap HP AFC column (from Amersham Biosciences) was pre-balanced with the binding buffer (10 mM phosphate buffer, pH 7.3, 140 mM NaCl, 2.7 mM KCl). GST or GST fusion protein solubilized in 8 M urea (60 μl) was loaded onto the column. The column was then washed with the binding buffer at each selected flow rate. Bound protein was eluted using the elution buffer (50 mM Tris–HCl, pH 8.0, 10 mM reduced glutathione). |
| Refolding Assay | Unspecified,Mass spectrometry,Reporter assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | |