Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cytosolic phospholipase A2 |
| Abbreviated Name | cPLA2 |
| SCOP Family | PLC-like (P variant) |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P47712 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Truncated C2 domain aa.1-178 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 178 |
| Molecular Weight | 20678.7 |
| Pi | 5.3888 |
| Molecular Weight | 20678.7 |
| Disulphides | 0 |
| Full Sequence |
MSFIDPYQHI IVEHQYSHKF TVVVLRATKV TKGAFGDMLD TPDPYVELFI STTPDSRKRT RHFNNDINPV WNETFEFILD PNQENVLEIT LMDANYVMDE TLGTATFTVS SMKVGEKKEV PFIFNQVTEM VLEMSLEVCS CPDLRFSMAL CDQEKTFRQQ RKEHIRESMK KLLGPKNS
|
| Notes | Other versions of truncated C2 domain expressed and refolded: aa.1-126 MSFIDPYQHI IVEHQYSHKF TVVVLRATKV TKGAFGDMLD TPDPYVELFI 50 STTPDSRKRT RHFNNDINPV WNETFEFILD PNQENVLEIT LMDANYVMDE 100 TLGTATFTVS SMKVGEKKEV PFIFNQ Number of amino acids: 126 Molecular weight: 14594.5 Theoretical pI: 4.93 aa.1-138 MSFIDPYQHI IVEHQYSHKF TVVVLRATKV TKGAFGDMLD TPDPYVELFI 50 STTPDSRKRT RHFNNDINPV WNETFEFILD PNQENVLEIT LMDANYVMDE 100 TLGTATFTVS SMKVGEKKEV PFIFNQVTEM VLEMSLEV Number of amino acids: 138 Molecular weight: 15956.1 Theoretical pI: 4.68 aa.1-155 MSFIDPYQHI IVEHQYSHKF TVVVLRATKV TKGAFGDMLD TPDPYVELFI 50 STTPDSRKRT RHFNNDINPV WNETFEFILD PNQENVLEIT LMDANYVMDE 100 TLGTATFTVS SMKVGEKKEV PFIFNQVTEM VLEMSLEVCS CPDLRFSMAL 150 CDQEK Number of amino acids: 155 Molecular weight: 17884.4 Theoretical pI: 4.66 |
| Expression | |
|---|---|
| Report | Nalefski EA, McDonagh T, Somers W, Seehra J, Falke JJ, Clark JD (1998) J Biol Chem, 273, 1365-1372 |
| Project Aim | Structure-Function |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | HB101 |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pTrcHisB |
| Expression Protocol | Cells were grown to late log phase and induced with 1mM IPTG and grown at 37degC. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Microfluidizer |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M GdnHCl |
| Refolding Buffer | 0.5M arginine HCl, 25mM Tris pH 8.0, 5mM DTT |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | DTT |
| Redox Agent Concentration | 0.5mM,0.5mM |
| Refolding Protocol | Harvested cells were lysed by microfluidization and inclusion bodies were collected. the Inclusion bodies were then washed and exracted with chloroform:methanol (2:1) to remove lipids. Inclusion bodies were then solubilized in solubilization buffer and passed over a TSK3000 gel filtration column (Toso-Haas) run in 8M urea, 20mM Tris pH 7.4, 5mM EDTA, 5mM DTT at room temperature. The selected peak was then passed over a MonoQ column at room temperature and eluted with 400-500mM NaCl. Selected fractions were refolded by dilution into refolding buffer while stirring slowly at 4degC. Refolded protein was then diluted slowly 2-fold into 25mM Tris pH 8, 5mM DTT and then again 2-fold into 2M (NH4)2SO4, 20mM Tris pH 8.0, 5mM DTT, 5mM EDTA. Refolded protein was then purified further using a Toyo-phenyl 650S column, a Mono Q column and a phenyl-5PW column. |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.5M |
| Refolding Yield | |
| Purity | |
| Notes | |