Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cerastocytin also known as Proaggregant serine proteinase |
| Abbreviated Name | CC-PPP |
| SCOP Family | Eukaryotic Proteases |
| Structure Notes | |
| Organism | Cerastes cerastes (Horned desert viper) |
| UniProt Accession | Q7SYF1 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 232 |
| Molecular Weight | 25386.0 |
| Pi | 8.46 |
| Molecular Weight | 25386.0 |
| Disulphides | 5 |
| Full Sequence |
VIGGAECNINEHRSLVLLYNSSRLFGGGTLINKEWVLSAAHCDGENMKIYLGLHHFRLPNKDRQIRVAKEKYFCRDRKSIVDKDIMLIKLNKPVNNSTHIAPLSLPSSPPSVGSDCRIMGWGTITSPNDTYPKVPHCANINILEHSLCERAYNDLSASSRTLCAGIEKGGIDTCKGDSGGPLICNGQIQGIVSWGDEVCGKPNKPGVYTKVFDYTDWIRNIIAGNTAATCPQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Dekhil H, Wisner A, Marrakchi N, El Ayeb M, Bon C, Karoui H. (2003) Biochemistry, 42, 10609-10618 |
| Project Aim | Cloning and expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 37.0 |
| Expression Time | 00 |
| Expression Vector | pET-17b |
| Expression Protocol | Expression and Folding of rCC-PPP. The expression and folding of rCC-PPP were performed according to the method of Zhang et al. (16), with some modifications. The sample was dialyzed, at 4 °C for 150 min, against 20 mM Tris-HCl (pH 8.0) containing 0.25 M NaCl, 2 mM EDTA, and 2 M urea. Bovine factor Xa was added at an enzyme:substrate ratio of 1:100, and digestion was conducted overnight at 37 °C. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 20 mM Tris-HCl (pH 9.5) containing 8 M urea, 2 mM EDTA, and 50 mM B-mercaptoethanol |
| Refolding Buffer | 20 mM Tris-HCl (pH 9.5) containing 2 mM EDTA |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 9.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The protein was then fully denatured by incubation for 120 min at room temperature in 20 mM Tris-HCl (pH 9.5) containing 8 M urea, 2 mM EDTA, and 50 mM B-mercaptoethanol. The refolding was started by diluting the sample 50-fold in 20 mM Tris-HCl (pH 9.5) containing 2 mM EDTA. Then the refolding process was allowed to proceed at room temperature under constant agitation. It was monitored by assaying the amidolytic activity of rCC-PPP with the chromogenic substrate S-2238. When the enzyme activity reached a maximal level, usually after 30-48 h, the refolding mixture was stored at 4 °C for subsequent purification. |
| Refolding Assay | Kinetic analysis,Computer analysis |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |