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 | 366 |
| Molecular Weight | 40608.7 |
| Pi | 4.82 |
| Molecular Weight | 40608.7 |
| Disulphides | 3 |
| Full Sequence |
MAEIT 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 350
SQKWILGDVF IREYYSVFDR ANNLVGLAKA I
|
| Notes | Prochymosin B allele: Gly244 Prochymosin A allele: Asp244, same results |
| Expression | |
|---|---|
| Report | Menzella HG, Gramajo HC, Ceccarelli EA (2002) Protein Expression and Purification, 25, 248-255 |
| 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 | 6h |
| Expression Vector | pET22b |
| Expression Protocol | Cells were grown in LB medium containing ampicillin (100microg/ml) at 37degC until A600 reached 0.7. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.7 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 1:10mM EDTA pH8.0, 0.5%(v/v)Triton X-100, 2:20mM KH2PO4 |
| Solubilization Buffer | 8M urea, 50mM KH2PO4 pH 10.5 |
| Refolding Buffer | 50mM KH2PO4 pH 10.5, various additives including Cu2+, L-arginine, CHAPS, Triton X-100 (see notes) |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 10.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.8mg/ml |
| Refolding Time | 13-16h |
| Redox Agent | Cu2+ |
| Redox Agent Concentration | 10microM |
| Refolding Protocol | 10g harvested cells were resuspended in 40ml of 50mM TrisHCl pH 8.0 and incubated at 37degC for 30min with 0.2mg/ml lysozyme. The mixture was sonicated on ice then centrifuged (10000g, 20min, 20degC), washed twice with 50ml wash buffer 1 and once with wash buffer 2. The inclusion bodies were then resuspended in refolding buffer to a final concentration of 20mg/ml. The solution was incubated with agitation for 2h at 30degC and centrifuged (10000g, 10min, 20degC). The protein was folded by dilution of 40ml unfolded protein solution in 1L of refolding buffer. Refolding was performed at 4degC for 13-16h. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | various |
| Refolding Yield | up to 67% |
| Purity | n/a |
| Notes | Various refolding conditions investigated, with NaCl, L-arginine, Tris, glycerol, PEG3500, CHAPS, Triton X-100, CTAB, SDS, GSH/GSSG, DTT, Cu2+. Most effective renaturation with 10microM Cu2+ and air oxidation - 48% recovery. Addition of 0.5M L-arginine, 1mg/L CHAPS or 0.1mg/L Triton X-100 increased yield to 67, 65, 60% respectively |