Refolding Record:
| Protein | |
|---|---|
| Protein Name | Phospholipase C |
| Abbreviated Name | PLC |
| SCOP Family | Phospholipase C |
| Structure Notes | |
| Organism | Bacillus cereus |
| UniProt Accession | P09598 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 287 |
| Molecular Weight | 32383.3 |
| Pi | 7.13421 |
| Molecular Weight | 32383.3 |
| Disulphides | 0 |
| Full Sequence |
MKKKVLALAAAITVVAPLQSVAFAHENDGGSKIKIVHRWSAEDKHKEGVNSHLWIVNRAI
DIMSRNTTLVKQDRVAQLNEWRTELENGIYAADYENPYYDNSTFASHFYDPDNGKTYIPF
AKQAKETGAKYFKLAGESYKNKDMKQAFFYLGLSLHYLGDVNQPMHAANFTNLSYPQGFH
SKYENFVDTIKDNYKVTDGNGYWNWKGTNPEEWIHGAAVVAKQDYSGIVNDNTKDWFVKA
AVSQEYADKWRAEVTPMTGKRLMDAQRVTAGYIQLWFDTYGDR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Tan CA, Hehir MJ, Roberts MF. (1997) Protein Expression and Purification, 10, 365-372 |
| Project Aim | Structure-Function |
| 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 | 2-3h |
| Expression Vector | pET23a |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 50mM Tris-HCl, 2mM EDTA, pH 8.0 |
| Solubilization Buffer | 8M urea, 25mM barbital, pH 7.5 |
| Refolding Buffer | 1.5 M urea, 25mM barbital, 0.1mM ZnSO4 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The inclusion bodies were denatured with 20 ml of buffer containing 8 M urea and 25 mM barbital buffer, pH 7.5 (adjusted by acetic acid). The solution was stirred at room temperature for at least 1 h or until the sample turned clear. The amount of urea was gradually adjusted to 4 M by addition of 25 mM barbital buffer, pH 7.5, and the sample was stirred overnight at 47C. Refolding was initiated by addition of zinc to a final concentration of 0.1 mM, and the stirring was continued for another 2 h. The protein was dialyzed against 1.5 M urea in 25 mM barbital, pH 7.5, and 0.1 mM ZnSO4 (refolding buffer). Clostripain was added to the dialyzed protein in a clostripain-to-inclusion body ratio of 1:50 (w/w), with 10 mM dithiothreitol added to activate clostripain. The sample was incubated at 377C for 20 min followed by heating at 50C for 20 min. The remaining urea was gradually removed by dialysis first against 0.3 M urea in refolding buffer and finally against refolding buffer without urea. The cleavage of the signal peptide in the presence of 1.5M urea was vital - cleavage of the peptide in lower concentrations of urea resulted in precipitation. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 50% |
| Purity | single band on SDS gel |
| Notes | |