Refolding Record:
| Protein | |
|---|---|
| Protein Name | Hemagglutinin |
| Abbreviated Name | FHA2 |
| SCOP Family | Influenza hemagglutinin (stalk) [58065] |
| Structure Notes | |
| Organism | Influenza A virus |
| UniProt Accession | P03437 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Trimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 221 |
| Molecular Weight | 25508.9 |
| Pi | 5.13 |
| Molecular Weight | 25508.9 |
| Disulphides | 1 |
| Full Sequence |
GLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAEEMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGFIMWACQRGNIRCNICI
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Curtis-Fisk J, Spencer RM, Weliky DP. (2008) Protein Expression and Purification, 1, 1 |
| Project Aim | Over expression & Renaturation |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | n/a |
| Expression Protocol | FHA2 expression was induced by addition of isopropyl thiogalactoside (IPTG) to a final concentration of 0.2 mM. For production of FHA2 with 1-13C, 15N Leu isotopic labeling, 100 mg/L of labeled amino acid was added at the time of protein induction. FHA2 production was continued for 3 h at 37 °C. The cell pellet was harvested by centrifugation at 10,000g for 10 min, and the pellet was then stored at −80 °C until purification. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM sodium phosphate at pH 8.0, 300 mM NaCl, 20 mM imidazole, and 0.5% (w/v) N-lauroylsarcosine (Sarkosyl) |
| Solubilization Buffer | 8 M urea, 100 mM NaH2PO4, 10 mM Tris–Cl |
| Refolding Buffer | 1 M arginine, 10 mM Tris–Cl, 0.17% decyl maltoside, 2 mM EDTA at pH 8 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The inclusion body fraction of FHA2 was defined as the component which pelleted during the centrifugation of the cell lysate. The pellet was sonicated in denaturing lysis buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris–Cl), the suspension centrifuged, and the supernatant purified with chelated cobalt His-Select resin using methods similar to those described above. After binding the denatured protein to the resin, the column was washed with 3 column volumes of denaturing wash buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris–Cl, 20 mM imidazole) and the FHA2 was eluted with 5 column volumes of denaturing elution buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris–Cl, 250 mM imidazole). The denatured FHA2 solution was rapidly diluted into twice the volume of ice cold refolding buffer (1 M arginine, 10 mM Tris–Cl, 0.17% decyl maltoside, 2 mM EDTA at pH 8) and stored at 4 °C overnight. Removal of urea and refolding of FHA2 was achieved with dialysis at 4 °C for two days using 10,000 MWCO tubing and a dialysis buffer [7]. |
| Refolding Assay | Circular Dichroism (uv-CD) |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 1 M |
| Refolding Yield | 10 mg/L |
| Purity | n/a |
| Notes | n/a |