Refolding Record:
| Protein | |
|---|---|
| Protein Name | C epsilon 3 |
| Abbreviated Name | Ce3 |
| SCOP Family | C1 set domains (antibody constant domain-like) |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01854 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | third constant domain of Immunoglobulin Epsilon heavy chain |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 131 |
| Molecular Weight | 14432.3 |
| Pi | 10.2 |
| Molecular Weight | 14432.3 |
| Disulphides | 1 |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSHMSADSNPRGVSAYLSRPSPFDL FIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEE KQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRST SKTS
|
| Notes | Gene cloned in pET 28 vector, expressed in BL21 cells |
| Expression | |
|---|---|
| Report | Unpublished (0) J Virol Methods, 0, 0 |
| Project Aim | Biophysical Studies |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | E.coli |
| Expression Strain | BL 21 (DE3) |
| Expression Temp | 37.0 |
| Expression Time | 8 |
| Expression Vector | pET28 |
| Expression Protocol | The expression of Ce3 is carried out by setting and overnight culture of 10 ml from a single colony which is then used to inoculate large volume culture. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.8 = n/a |
| Cell Disruption Method | Cell disrupter |
| Lytic Agent | Chemicals |
| Pre-Refolding Purification | Ni-NTA column |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: affinity immobilization |
| Wash Buffer | 100mM EDTA, 1mM DTT, 1% triton -X100, 1mM PMSF, 50mM MgCl2, pH 7.4 |
| Solubilization Buffer | 6M GnHCl, 20 mM Phosphate, 0.5M NaCl pH 7.4 |
| Refolding Buffer | 20 mM Phosphate, .5 M NAcl pH 7.4 |
| Pre-Refolding Purification | Ni-NTA column |
| Tag Cleaved | no |
| Refolding pH | 7.4 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 1mg to 5 mg/ml |
| Refolding Time | one hour |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Refolding of this protein is carried out using a 3 buffer system. the denatured and reduced protein is present in sloubilization buffer (6M GnHCl, 20mM Phosphate and 0.5M NaCl, 1mM DTT) which is loaded on nickle colum pre-equlibrated with 6M GnHCl, 20mM Phosphate and 0.5M NaCl pH 7.4. after immobilizing Ce3 on Ni column, the gradient of 1 ml/min is created to change the buffer to refolding buffer (20mM Phosphate and 0.5M NaCl pH 7.4)over period of one hour. the protein is eluted by gradient of elution buffer (20mM Phosphate and 0.5M NaCl pH 1.5) |
| Refolding Assay | 1H chemical shift (ppm) |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 0% |
| Purity | >90% |
| Notes | The eluted protein is free of impurities and is monomeric, but its not folded so it aggregates over a very short period of time. the aggrgation increases if the protein concentration is high (3mg/ml) |