Refolding Record:
| Protein | |
|---|---|
| Protein Name | Apolipoprotein C-II |
| Abbreviated Name | Apo-CII |
| SCOP Family | Fragments of apolipoproteins |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | O95996 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Membrane and cell surface proteins and peptides |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 102 |
| Molecular Weight | 11283.9 |
| Pi | 4.72187 |
| Molecular Weight | 11283.9 |
| Disulphides | 0 |
| Full Sequence |
MGTRLLPALFLVLLVLGFEVQGTQQPQQDEMPSPTFLTQVKESLSSYWESAKTAAQNLYE
KTYLPAVDEKLRDLYSKSTAAMSTYTGIFTDQVLSVLKGEE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hatters DM, MacPhee CE, Lawrence LJ, Sawyer WH, Howlett GJ. (2000) Biochemistry, 39, 8276-8283 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | unknown |
| Expression Vector | pET11a |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 6 mM urea, 10 mM TrisHCl, pH 8.0 |
| Solubilization Buffer | 5 mM GdnHCl, 100 mM arginine, pH 12.0 |
| Refolding Buffer | 100mM urea and 10mM sodium phosphate |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.4 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 50 micrograms/mL |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | As misfolding induces aggregation of recombinant proteins , apoC-II was refolded at a relatively low concentration and add 100 mM urea to the buffers after the protein was refolded to inhibit intermolecular hydrophobic interactions. A stock of apoC-II at 9.5 mg/mL in 5 M guanidine HCl was diluted directly into 100 mL of 100 mM sodium phosphate, pH 7.4, to a final protein concentration of 50 microg/mL. The protein was incubated at room temperature for 3 h and then dialyzed into 100 mM urea and 10 mM sodium phosphate, pH 7.4, at 4 C with two buffer changes. The protein solution was applied to a HR5-10 column (Pharmacia-Biotech) packed with Source 15Q anion-exchange resin (Pharmacia-Biotech) preequilibrated in 100 mM urea, 10 mM sodium phosphate, pH 7.4. The protein was eluted with the above buffer and 500 mM NaCl at a flow rate of 1 mL/min. The eluted protein was desalted by application to a 1 ?9 cm column of Sephadex G-25 resin preequilibrated with 100 mM sodium phosphate, pH 7.4, and eluted at a flow rate of 1 mL/min. ApoC-II prepared by this method and used directly is referred to as freshly prepared. |
| Refolding Assay | Near-UV Circular Dichroism |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | |