Refolding Record:
| Protein | |
|---|---|
| Protein Name | Profilin II |
| Abbreviated Name | Profilin II |
| SCOP Family | Profilin (actin-binding protein) |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P35080 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 140 |
| Molecular Weight | 15046.2 |
| Pi | 6.54546 |
| Molecular Weight | 15046.2 |
| Disulphides | 0 |
| Full Sequence |
MAGWQSYVDNL MCDGCCQEAA IVGYCDAKYV WAATAGGVFQ SITPIEIDMI VGKDREGFFT NGLTLGAKKC SVIRDSLYVD GDCTMDIRTK SQGGEPTYNV AVGRAGRVLV FVMGKEGVHG GGLNKKAYSM AKYLRDSGF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Sharma A, Lambrechts A, Hao le T, Le TT, Sewry CA, Ampe C, Burghes AH, Morris GE (2005) Experimental Cell Research, 309, 185-197 |
| Project Aim | Functional Studies |
| Fusion | None |
| 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 | 3-4h |
| Expression Vector | pET11d |
| Expression Protocol | Cells incubated at 37degC were induced with 1mM IPTG and grown for a further 3-4h . |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20mM TrisHCl pH 8.1, 1mm EDTA, 5mM DTT |
| Solubilization Buffer | 20mM TrisHCl pH 8.1, 1mm EDTA, 5mM DTT, 8M urea |
| Refolding Buffer | 20mM TrisHCl pH 8.1, 1mm EDTA, 5mM DTT |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.1 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 5mM,5mM |
| Refolding Protocol | Cell pellets were resuspended in 20mM TrisHCl pH 8.1, 1mM EDTA, 5mM DTT supplemented with prtease inhibitors. Cells were then lysed by French press and the lysate was centrifuged (100000g). The supernatant was loaded on a poly(L-proline)-sepharose column. The column was washed with the same buffer, then the protein were eluted with solubilization buffer. The protein was then refolded by step-wise dialysis in refolding buffer with sequentially decreasing amounts of urea. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |