Refolding Record:
| Protein | |
|---|---|
| Protein Name | 3,5-cyclic phosphodiesterase B |
| Abbreviated Name | PDE3B |
| SCOP Family | PDEase |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q13370 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 421 |
| Molecular Weight | 48236.1 |
| Pi | 4.91 |
| Molecular Weight | 48236.1 |
| Disulphides | 0 |
| Full Sequence |
MEQEVSLD LILVEEYDSL IEKMSNWNFP IFELVEKMGE KSGRILSQVM YTLFQDTGLL EIFKIPTQQF MNYFRALENG YRDIPYHNRI HATDVLHAVW YLTTRPVPGL QQIHNGCGTG NETDSDGRIN HGRIAYISSK SCSNPDESYG CLSSNIPALE LMALYVAAAM HDYDHPGRTN AFLVATNAPQ AVLYNDRSVL ENHHAASAWN LYLSRPEYNF LLHLDHVEFK RFRFLVIEAI LATDLKKHFD FLAEFNAKAN DVNSNGIEWS NENDRLLVCQ VCIKLADING PAKVRDLHLK WTEGIVNEFY EQGDEEANLG LPISPFMDRS SPQLAKLQES FITHIVGPLC NSYDAAGLLP GQWLEAEEDN DTESGDDEDG EELDTEDEEM ENNLNPKPPR RKSRRRIFCQ LMHHLTENHK IWK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chung C, Varnerin JP, Morin NR, MacNeil DJ, Singh SB, Patel S, Scapin G, Van der Ploeg L, Tota MR (2003) Biochem Biophys Res Commun., 307, 1045-1050 |
| Project Aim | Structure-Function |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | CodonPlus-RIL |
| Expression Temp | 37.0 |
| Expression Time | 2h |
| Expression Vector | pET30a |
| Expression Protocol | Cells were grown at 37degC until OD600 reached 1.0, then expression was induced with 1mM IPTG. Cells were harvested after 2h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 1.0 |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris pH 7.5, 300mM NaCl, 1:1000 protease inhibitors (Sigma P-8849), 0.5% Triton X-100 OR 20mM EDTA |
| Solubilization Buffer | 50mM Tris pH 8.0, 7M GdnHCl, 0.1M DTT |
| Refolding Buffer | 630mM Tris pH 7.5, 30% glycerol, 20mM MgCl2, 40microM ZnSO4, 0.02% NaN3, 0.1mM PMSF, 1:1000 protease inhibitors, 10mM DTT, 0.5M arginine, 5mM MnCl2, 10mM NaCl, 0.5mM KCl |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 48-72h |
| Redox Agent | DTT |
| Redox Agent Concentration | 10mM,10mM |
| Refolding Protocol | Cells were lysed with 0.1mg/ml lysozyme by freeze-thawing and treated with DNase1. The inclusion bodies were isolated by centrifugation and washed with wash buffer containing Triton X-100, followed by wash buffer with EDTA. The inclusion bodies were then dissolved in solubilization buffer for 2h at 4degC. The protein was refolded by dilution into refolding buffer for 48-72h at 4degC with no agitation |
| Refolding Assay | enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine,Glycerol |
| Additives Concentration | 30%,.5M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |