Refolding Record:
| Protein | |
|---|---|
| Protein Name | Kallikrein-4/Prostase |
| Abbreviated Name | Kallikrein-4/Prostase |
| SCOP Family | Eukaryotic Proteases |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q9Y5K2 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | Propiece of hK4 was replaced by that of prostate-specific antigen (PSA) to create an activation site susceptible to trypsin-type proteases |
| Variants | n/a |
| Chain Length | 233 |
| Molecular Weight | 24459.6 |
| Pi | 4.70841 |
| Molecular Weight | 24459.6 |
| Disulphides | Unknown |
| Full Sequence |
GSCSQIINGEDCSPHSQPWQAALVMENELFCSGVL
VHPQWVLSAAHCFQNSYTIGLGLHSLEADQEPGSQMVEASLSVRHPEYNRPLLANDLMLI
KLDESVSESDTIRSISIASQCPTAGNSCLVSGWGLLANGRMPTVLQCVNVSVVSEEVCSK
LYDPLYHPSMFCAGGGQDQKDSCNGDSGGPLICNGYLQGLVSFGKAPCGQVGVPGVYTNL
CKFTEWIEKTVQAS
|
| Notes | hK4 (prostase, KLK4), a recently cloned prostate-specific serine protease and a member of the tissue kallikrein family, is a zymogen composed of 228 amino acid residues including an amino-terminal propiece, Ser-Cys-Ser-Gln-. A chimeric form of hK4 (ch-hK4) was constructed in which the propiece of hK4 was replaced by that of prostate-specific antigen (PSA) to create an activation site susceptible to trypsin-type proteases. |
| Expression | |
|---|---|
| Report | Takayama TK, McMullen BA, Nelson PS, Matsumura M, Fujikawa K. (2001) Biochemistry, 40, 15341-15348 |
| Project Aim | Undefined |
| 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 | 2h |
| Expression Vector | pET12a |
| Expression Protocol | The majority of procedures were described previously (14). The pET12-proPSA-hK4 chimera plasmid was constructed as above. Escherichia coli strain BL21(DE3) was transformed, the protein was expressed, and the inclusion bodies were isolated and solubilized by the previously published methods (14) with some modifications. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | Not stated |
| Solubilization Buffer | 8 M urea, 50 mM Tris-HCl, pH 8.5, 150 mM NaCl, 0.1 M NH4Cl, 2 mM EDTA, 10 mM benzamidine, 0.1 M 2-mercaptoethanol, 1 mM diisopropyl fluorophosphate |
| Refolding Buffer | 2M urea, 0.5M NaCl, 0.1M NH4Cl, 1mM EDTA, 10mM benzamidine, 1.25mM GSH, 0.5mM GSSG, 50mM Tris-HCl |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.8 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.003mg/ml |
| Refolding Time | 24h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a |
| Refolding Protocol | 8 M urea, 50 mM Tris-HCl, pH 8.5, 150 mM NaCl, 0.1 M NH4Cl, 2 mM EDTA, 10 mM benzamidine, 0.1 M 2-mercaptoethanol, 1 mM diisopropyl fluorophosphate by shaking for 4 h at room temperature. After a small amount of insoluble material was removed by centrifugation (10,000 ?g for 5 min), the solubilized protein (25 mg) was then refolded by diluting into 8 liters of 2 M urea, 50 mM Tris-HCl, pH 8.8, 0.5 M NaCl, 0.1 M NH4Cl, 1 mM EDTA, 10 mM benzamidine, 1.25 mM reduced glutathione, 0.5 mM oxidized glutathione and by slowly stirring for 24 h at 4 °C. The final protein concentration was ~0.003 mg/ml. The refolding buffer was degassed and flushed under N2 gas prior to use. The sample was then dialyzed against 65 liters of 10 mM Tris-HCl, pH 8.0, with three buffer changes. Upon refolding, the protein self-processed from a 25kDa precursor to a 21kDa active enzyme. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 25% |
| Purity | |
| Notes | |