Refolding Record:
| Protein | |
|---|---|
| Protein Name | Trypsinogen |
| Abbreviated Name | Trypsinogen |
| SCOP Family | Eukaryotic Proteases |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P07477 |
| 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 | 247 |
| Molecular Weight | 26558.1 |
| Pi | 6.08 |
| Molecular Weight | 26558.1 |
| Disulphides | 5 |
| Full Sequence |
MNPLLILTFVAAALAAPFDDDDKIVGGYNCEENSVPYQVSLNSGYHFCGGSLINEQWVVS
AGHCYKSRIQVRLGEHNIEVLEGNEQFINAAKIIRHPQYDRKTLNNDIMLIKLSSRAVIN
ARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKIT
SNMFCVGFLEGGKDSCQGDSGGPVVCNGQLQGVVSWGDGCAQKNKPGVYTKVYNYVKWIK
NTIAANS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Buswell, M. A., Ebtinger, M., Vertes, A. A., Middelberg, A. P. J. (2002) Biotechnology and Bioengineering, 77, 435-444 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | not stated |
| Expression Protocol | Inclusion bodies were isolated from E.coli cells. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 7M urea, 100mM cysteine, 10mM EDTA, pH 9.5 |
| Refolding Buffer | 50mM CaCl2, 5mM Tris, 3mM cysteine, 1mM cystine, pH 9.0 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 9.0 |
| Refolding Temperature | 8.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Cysteine/Cystine |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Inclusion bodies underwent extensive washing. First they were centrifuged(18,000g, 55min) and supernatant decanted. The resulting pellet was resuspended in 20mM Tris, 50mM NaCl, pH 8.0 and centrifuged again(18,000, 25min). The resulting pellet was resuspended in 20mM Tris, 5mM EDTA, pH 8.0. Lysozyme was added to the mixture to a final concentration of 0.1%(w/v) and the suspension incubated at room temperature for 60min before centrifugation(18,000g, 10min). The pellet was then resuspended in 20mM Tris, 5mM EDTA, pH 8.0 and Triton X-100 added to 1% (v/v) and the suspension mixed for 10min. The suspension was then centrifuged (18,000g, 15min). The pellet was resuspended in 20mM Tris at pH 7.5 and then centrifuged. This process was repeated a further two times, but on last resuspension the buffer was pH 8.0. The suspension was then dialyzed twice against 10mM Tris at pH 8.0 for 12h at 4degC and then centrifuged(9,000g, 15min) and inclusion bodies frozen. Inclusion bodies were solubilized for between 30 and 60min. They were then refolded in a 100ml nonbaffled glass reaction vessel at 8degC. In each case refolding started by the injection of solubilized inclusion body into 100ml of refolding buffer. Following the initial injection there were five others, each 40min apart. After the six injections the solution was maintained for 40min. |
| Refolding Assay | Folding Kinetics |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |