Refolding Record:
| Protein | |
|---|---|
| Protein Name | Prochymosin |
| Abbreviated Name | Prochymosin |
| SCOP Family | Pepsin-like proteases |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | P00794 |
| 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 | 322 |
| Molecular Weight | 35595.9 |
| Pi | 4.41724 |
| Molecular Weight | 35595.9 |
| Disulphides | 3 |
| Full Sequence |
EVASVPLTNY LDSQYFGKIY LgtppQEFTV LFDTGSSDFW VPSIYCKSNA CKNHQRFDPR KSSTFQNLGK PLSIHYGTGS MQGILGYDTV TVSNIVDIQQ TVGLSTQEPG DVFTYAEFDG ILGMAYPSLA SEYSIPVFDN MMNRHLVAQD LFSVYMDRNG QESMLTLGAI DPSYYTGSLH WVPVTVQQYW QFTVDSVTIS GVVVACEGGC QAILDTGTSK LVGPSSDILN IQQAIGATQN QYGEFDIDCD NLSYMPTVVF EINGKMYPLT PSAYTSQDQG FCTSGFQSEN HSQKWILGDV FIREYYSVFD RANNLVGLAK AI
|
| Notes | 3 mutants created - substituting in for GTPP (aa.22-25) GG/GS/SG For all three mutants: Number of amino acids: 320 Molecular weight: 35357 Theoretical pI: 4.42 |
| Expression | |
|---|---|
| Report | Li H, Zhang Y, Dong Y, Yang K (1998) Biochim Biophys Acta., 1384, 121-129 |
| Project Aim | Structure-Function |
| Fusion | pro sequence |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 42.0 |
| Expression Time | not stated |
| Expression Vector | pBC4 |
| Expression Protocol | Expression of protein was induced by a shift of growth temperature from 30degC to 42degC. |
| Method of Induction | Temperature Shift |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | N/A |
| Solubilization Buffer | 50mM KH2PO4, 1mM EDTA, 50mM NaCl, pH 11, 8M Urea |
| Refolding Buffer | 1: 50mM KH2PO4, 1mM EDTA, 50mM NaCl, pH 11, 2: 20mM TrisHCl pH 8.0, 1mM EDTA, 50mM NaCl |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | overnight |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Inclusion bodies obtained from lysed cells were suspended in solubilization buffer to give a protein concentration of 2mg/ml. After incubation at 30degC for 2h, the solution was centrifuged (10000g, 10min). The supernatant was diluted 10-fold with refolding buffer 1 and left at 15degC for 2h. The sthe solution was adjusted to 8.0 with 1N HCl, maintained for 1h and dialyzed against refolding buffer 2 at 4degC overnight. The protein was then activated at pH 2 and 15-20degC for 2-14h to produce pseudochymosin, which was then dialysed against 0.1M sodium acetate pH 5.5 at 25degC overnight to facilitate activation of the protein |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |