Refolding Record:
| Protein | |
|---|---|
| Protein Name | Acutin |
| Abbreviated Name | Acutin |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Agkistrodon acutus |
| UniProt Accession | Q9YGS1 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 242 |
| Molecular Weight | 26472.9 |
| Pi | 5.90096 |
| Molecular Weight | 26472.9 |
| Disulphides | 6 |
| Full Sequence |
MVIGGDECDI NEHRFLVAFF NTTGFFCGGT LINPEWVVTA AHCDSTNFQM QLGVHSKKVL NEDEQTRNPK EKFICPNKNN NEVLDKDIML IKLDKPISNS KHIAPLSLPS SPPSVGSVCR IMGWGSITPV KETFPDVPYC ANINLLDHAV CQTGYPSCWR NTTLCAGFLE GGKDTCGGDS GGPLICNGQF QGIVSYGAHS CGQGPKPGIY TNVFDYTDWI QRNIAGNTDA TCPP LEHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Pan H, Du X, Yang G, Zhou Y, Wu X (1999) Biochemical and Biophysical Research Com, 255, 412-415 |
| Project Aim | Structure-Function |
| Fusion | C-terminal hexahistidine tag |
| 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 | 4h |
| Expression Vector | pET24a |
| Expression Protocol | 500ml of 2xYT culture was inoculated with 1:50 volume overnight culture. The culture was induced with IPTG when A600nm reached 0.6. The culture was then incubated for a further 4h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | not stated |
| Solubilization Buffer | 6M urea, 10mM EDTA, 10mM DTT |
| Refolding Buffer | 50mM TrisHCl, 20% glycerol, 0.5M NaCl, 0.1mM EDTA, 8mM L-cysteine, 1mM cystine |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 20h |
| Redox Agent | Cysteine/Cystine |
| Redox Agent Concentration | 8mM/1mM |
| Refolding Protocol | The protein was purified under denaturing conditions by Ni2+ IDA sepharose affinity column. The purified protein was then dialyzed in solubilization buffer for 2h, then dialyzed against refolding buffer at room temperature for 20h. When refolding was completed, the protein solution was dialyzed against 50mM Tris and then concentrated. |
| Refolding Assay | Amino acid sequencing |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | 5mg/L culture |
| Purity | 90% |
| Notes | |