Refolding Record:
| Protein | |
|---|---|
| Protein Name | Tobacco anionic peroxidase |
| Abbreviated Name | TOP |
| SCOP Family | CCP-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q40555 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 296 |
| Molecular Weight | 31737.1 |
| Pi | 4.37313 |
| Molecular Weight | 31737.1 |
| Disulphides | Unknown |
| Full Sequence |
MIPLALMLQV LYVVLWIKGN VLMLELVLKL FVFISMIVLL IGCDGSILLD TDGTQTEKDA PANVGAGGFD IVDDIKTALE NVCPGVVSCA DILALASEIG VVLAKGPSWQ VLFGRKDSLT ANRSGANSDI PSPFETLAVM IPQFTNKGMD LTDLVALSGA HTFGRARCGT FEQRLFNFNG SGNPDLTVDA TFLQTLQGIC PQGGNNGNTF TNLDISTPND FDNDYFTNLQ SNQGLLQTDQ ELFSTSGSAT IAIVNRYAGS QTQFFDDFVS SMIKLGNISP LTGTNGQIRT DCKRVN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hushpulian DM, Savitski PA, Rojkova AM, Chubar TA, Fechina VA, Skharov IY, Lagrimini LM, Tishov VI, Gazaryan IG (2003) Biochemistry (Moscow), 68, 1189-1194 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)CodonPlus |
| Expression Temp | 37.0 |
| Expression Time | 4-6h |
| Expression Vector | pET40b |
| Expression Protocol | Cells were grown in 400ml LB medium prepared with 10mM TrisHCl pH 8.0, containing 0.04mg/ml kanamycin. When OD600 reached 0.5-0.7, expression was induced by the addition of 0.2mM IPTG, and cells were grown for a further 4-6h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5-0.7 |
| Cell Disruption Method | Osmotic shock + sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 0.05M TrisHCl, pH 8.5 |
| Solubilization Buffer | 6M Urea, 1mM DTT |
| Refolding Buffer | 5mM CaCl2, 5microM hemin, 5% glycerol, 50mM TrisHCl pH 8.4, 1.8-2.3M urea, 0.1-0.7mM GSSH, 0.1-1mM DTT |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 6-7days |
| Redox Agent | GSSG/DTT |
| Redox Agent Concentration | 0.1-0.7mM/0.1-1mM |
| Refolding Protocol | Cells were collected by centrifugation (5000g) and resuspended in 10mM TrisHCl pH 8.0 and disrupted by sonication in the presence of 2M NaCl and 10mM DTT. The mixture was incubated for 1.5h at room temperature and then sonicatin was repeated. The insluble inclusion bodes were then washed with wash buffer and dissolved in 60ml solubilization buffer. The protein was then refolded by drop-ise addition of the protein to 600ml refolding buffer. Diffrent refolding buffers contained different concentrations of urea, GSSH and DTT. The activity of the protein was monitured during refolding, when activity reached a plateau, the mixture was saturated with 60% ammonium sulfate. The precipitate was centrifuged and dissolved in water. The resulting solution was purified using a Sephacryl S-200 column equilibrated with 5mM TrisHCl pH 8.5 |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 5% |
| Refolding Yield | |
| Purity | 85-90% |
| Notes | Maximum yield obtained in buffer with 1.9M Urea, pH 9.5, DTT:GSH ratio 1:7-1:5 |