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 | 381 |
| Molecular Weight | 42179.7 |
| Pi | 4.9 |
| Molecular Weight | 42179.7 |
| Disulphides | 3 |
| Full Sequence |
MRCLVVLLAV FALSQGAEIT RIPLYKGKSL RKALKEHGLL EDFLQKQQYG ISSKYSGFGE VASVPLTNYL DSQYFGKIYL GTPPQEFTVL FDTGSSDFWV PSIYCKSNAC KNHQRFDPRK SSTFQNLGKP LSIHYGTGSM QGILGYDTVT VSNIVDIQQT VGLSTQEPGD VFTYAEFDGI LGMAYPSLAS EYSIPVFDNM MNRHLVAQDL FSVYMDRNGQ ESMLTLGAID PSYYTGSLHW VPVTVQQYWQ FTVDSVTISG VVVACEGGCQ AILDTGTSKL VGPSSDILNI QQAIGATQNQ YGEFDIDCDN LSYMPTVVFE INGKMYPLTP SAYTSQDQGF CTSGFQSENH SQKWILGDVF IREYYSVFDR ANNLVGLAKA I
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chitpinityol S, Goode D, Crabbe MJC (1998) Molecular Vision, 4, 1-7 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 8h |
| Expression Vector | pET-3D |
| Expression Protocol | Cells were grown in LB broth with 100microg/ml ampicillin. When OD600 reached 0.6-1.0, expression was induced by the addition of 0.4mM IPTG. After a further 8h, cells were harvested by centrifugation (6500g, 10min) |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6-1.0 |
| Cell Disruption Method | Chemical |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Chaperone-assisted refolding |
| Wash Buffer | 50mM Tris pH 8.0, 10mM EDTA, 50mM NaCl, 0.1mM PMSF, 0.5% (v/v) Triton X-100 |
| Solubilization Buffer | 50mM Tris pH 8.0, 10mM EDTA, 50mM NaCl, 0.1mM PMSF, 0.5% (v/v) Triton X-100, 8M urea |
| Refolding Buffer | 1: 50mM KH2PO4, pH 10.7, 1mM EDTA, 50mM NaCl, alpha-crystallin 2:20mM TrisHCl, 50mM NaCl, 1mM EDTA pH 8.0 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 10.7 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.25mg/ml |
| Refolding Time | overnig |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were lysed with lysozyme and deoxycholate, then inclusion body pellets were washed in 9 volumes of wash buffer, incubated for 5min at room temperature, then centrifuged. The inclusion bodies were then dissolved in solubilization buffer and incubated at 25degC for 1h, then centrifuged. The solution was then diluted 25-fold in a high pH refolding buffer 1 containing alpha-crystallin. The solution was incubated at 25degC for 1h then adjusted to pH 8, incubation was then continued for 1h at 25degC. The solution was then dialyzed in refolding buffer 2 at 4degC. The folded protein was then activated by acidification to pH 2 (2h, 25degC), the pH was then adjusted to 6.3 (1h, 25degC). |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | alpha-crystallin |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |