Refolding Record:
| Protein | |
|---|---|
| Protein Name | A-protein |
| Abbreviated Name | A-protein |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Aeromonas salmonicida |
| UniProt Accession | P35823 |
| 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 | 502 |
| Molecular Weight | 53298.0 |
| Pi | 5.33935 |
| Molecular Weight | 53298.0 |
| Disulphides | Unknown |
| Full Sequence |
MDVVISPNDN TFVTTSLASV TKQPVLDFST AQQNLTLNFS EVGDLKNNGF IVLEIQGEGQ FNDAEIRQWL SNGFWRRPFT GLLVNPNDHG NFANSGEVND VRKFFKIISD GTQLTIVHTI DSNGKRLRLA LASDVEETIN FADAEVELKL NLANQAFKLT SGSQGTVALT AGALWNASYT ADPVATKPLF KLGKLFQLSL TNAGKATALV SEGFLKLNIG DANISATDFA ITNVTTNQTI QRDKVNLTLT GDVSAFKKDA NGNLVNKAGA SIGWKAAADG
QSATAVLGAG NMAGGVQNAL AAFGTLYVAA DNTVPVPAVN FNVKAEIQGD SQATYNYFKD ELADLFILTR DGMKFDTITT GTTSANLIHI RDVSNILPTE GGKIFVTITE YADHAANGRG EGTVLVTRKA LSVTLPSGGA VTLKPADVAA DVGASITAGR QARLVFEVET NQGEVAVKKS NAEGVDIQNG TRGTAPLVDF
TL RLLTKPERKLSWLLPPLSNN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Maurice S, Hadge D, Dekel M, Friedman A, Gertler A, Shoseyov O. (1999) Protein Expression and Purification, 16, 396-404 |
| Project Aim | Undefined,Recombinant Protein Expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 6h |
| Expression Vector | pET3d |
| Expression Protocol | 500ml culture of TB medium was inoculated with an overnight culture. Cells were grown until OD600 reached 0.9, expression with 0.4mM IPTG. Cells were harvested 6h later by centrifugation (6000g, 10 min) and stored at -20degC for later processing. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.9 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 0.1% Triton X-100 |
| Solubilization Buffer | 20mM Trizma base, 5mM betamercaptoethanol, pH 12 |
| Refolding Buffer | 10mM Tris-HCl |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 48h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Pelleted bacteria were suspended in 60 ml TrisHCl pH 8.0) with EDTA at a final concentration of 10 mM and lysozyme was added (0.5 mg/ml). The mixture was stirred (30 min, 4degC), followed by sonication and separation of soluble and insoluble fractions by centrifugation (25,000g, 30 min). Sedimented inclusion bodies were suspended in cold distilled water containing 0.1% Triton X-100, sonicated, and centrifuged repeatedly until all impurities were removed. This was followed by two more washes in water not containing Triton-X. Inclusion bodies were suspended by sonication in 20 mM Trizma Base containing 5 mM 2-mercaptoethanol 0.5 mg protein to 1 ml buffer) and the pH was adjusted 12 with NaOH. The clear refold solution was stirred h, 4degC) and dialyzed (48h) against 6x10 L of 10 mM TrisHCl (pH 8.0). |
| Refolding Assay | Immunoassay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 160mg/L culture |
| Purity | |
| Notes | |