Refolding Record:
| Protein | |
|---|---|
| Protein Name | Proinsulin |
| Abbreviated Name | Proinsulin |
| SCOP Family | Insulin-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01308 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 87 |
| Molecular Weight | 9394.7 |
| Pi | 5.20167 |
| Molecular Weight | 9394.7 |
| Disulphides | 3 |
| Full Sequence |
FVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAED
LQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bai Q, Kong Y, Geng X (2003) Chinese Chemical Letters, 14, 824-827 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DH5? |
| Expression Temp | 37.0 |
| Expression Time | 12h |
| Expression Vector | pBV 220 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: hydrophobic interaction chromatography |
| Wash Buffer | 50 mM PBS + 2.0 mol/L urea |
| Solubilization Buffer | 8.0 mol/L urea |
| Refolding Buffer | 80 mM Tris |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The sample solution of rh-proinsulin extracted with 8.0 mol?L-1 urea was directly injected into the USRPP and then eluted with an non-linear gradient elution by the mobile phases A[3.0 mol?L-1 (NH4)2SO4 + 0.08 mol?L-1 tris buffer (pH, 7.5)] and B[0.08 mol?L-1 tris buffer (pH, 7.5)]. Mobile phase for RPLC consisted of solutions A, 90% H2O + 10% CH3OH + 0.03% HCl and solution B, 10% H2O + 90% CH3OH + 0.03% HCl. Rh-proinsulin was enzyme-cleaved according to reference2. The rh-proinsulin concentration was detected according to the Bradford method |
| Refolding Assay | HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 80% |
| Purity | 90% |
| Notes | |