Refolding Record:
| Protein | |
|---|---|
| Protein Name | Self-incompatibility protein |
| Abbreviated Name | SI |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Papaver rhoeas |
| UniProt Accession | O65870 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 114 |
| Molecular Weight | 13858.3 |
| Pi | 6.46 |
| Molecular Weight | 13858.3 |
| Disulphides | Unknown |
| Full Sequence |
RFLPVIEVRIMNKRGNGHSIGIHCRSKDDDLGYHRISDGQQVHFSFRENFFHTTTFNCDIEWDSRRHFNFDSYRAQRDDHGRCTTECLWKTTDEGLYGYDQEHEYWQLYYLAKK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kakeda K, Jordan ND, Conner A, Ride JP, Franklin-Tong VE, Franklin FC. (1998) Plant Cell., 10, 1723-31 |
| Project Aim | Identification and Characterization |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 4 h |
| Expression Vector | pMS119 |
| Expression Protocol | Cells containing wild-type and mutant gene constructs were induced with 1 mM isopropyl 􏰉-D-thiogalactopyranoside for 4 hr, resulting in high-level expression of recombinant proteins that accumulated as insoluble inclusion bodies. Cells were harvested by centrifugation (5000g at 4􏰅C for 10 min) and resuspended in lysis buffer containing 50 mM Tris-Cl, 100 mM NaCl, and 1 mM EDTA, pH 8.0. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | Lysis buffer (50 mM Tris-Cl, 100 mM NaCl, and 1 mM EDTA, pH 8.0) |
| Solubilization Buffer | Lysis buffer containing 6 M guanidine–HCl, 0.5 M 2-mercaptoethylamine, and 0.125 mM phenylmethylsulfonyl fluoride, pH 8.0 |
| Refolding Buffer | 100 mM Tris containing 50 mM L-arginine, 2 mM EDTA, 10 mM cystamine, and 5% (v/v) glycerol, pH 8.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 14.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | Cystamine |
| Redox Agent Concentration | 10 mM |
| Refolding Protocol | They were disrupted by using lysozyme and deoxycholate and by sonication: inclusion bodies were collected by centrifugation and washed in lysis buffer (Sambrook et al., 1989). The resulting proteins were dissolved in lysis buffer containing 6 M guanidine–HCl, 0.5 M 2-mercaptoethylamine, and 0.125 mM phenylmethylsulfonyl fluoride, pH 8.0, for 1 hr at room temperature and refolded by rapid dilution in 100 mM Tris containing 50 mM L-arginine, 2 mM EDTA, 10 mM cystamine, and 5% (v/v) glycerol, pH 8.0, at 14􏰅C to a final concentration of 100 􏰊g/mL􏰃. After incubation overnight, the solution was dialyzed against 50 mM Tris-HCl and 100 mM NaCl, pH 8.0, and concentrated against the same buffer containing 12.5% polyethylene glycol 6000. The final protein solution was stored at 􏰃80􏰅C. Protein concentration was estimated using the Coomassie Brilliant Blue R 250 dye-binding method (Bradford, 1976). |
| Refolding Assay | Biological assay |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine,Glycerol |
| Additives Concentration | 5%(v/v), 50 mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |