Refolding Record:
| Protein | |
|---|---|
| Protein Name | 3,5-cyclic nucleotide phosphodiesterase 7A |
| Abbreviated Name | PDE7A1 |
| SCOP Family | Cyclic nucleotide phosphodiesterase |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q13946 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | catalytic domain (aa.147-482) |
| Chimera | n/a |
| Variants | Splice variant 1 (PDE7A1) |
| Chain Length | 344 |
| Molecular Weight | 39853.3 |
| Pi | 6.27502 |
| Molecular Weight | 39853.3 |
| Disulphides | 0 |
| Full Sequence |
MLEK VGNWNFDIFL FDRLTNGNSL VSLTFHLFSL HGLIEYFHLD MMKLRRFLVM IQEDYHSQNP YHNAVHAADV TQAMHCYLKE PKLANSVTPW DILLSLIAAA THDLDHPGVN QPFLIKTNHY LATLYKNTSV LENHHWRSAV GLLRESGLFS HLPLESRQQM ETQIGALILA TDISRQNEYL SLFRSHLDRG DLCLEDTRHR HLVLQMALKC ADICNPCRTW ELSKQWSEKV TEEFFHQGDI EKKYHLGVSP LCDRHTESIA NIQIGFMTYL VEPLFTEWAR FSNTRLSQTM LGHVGLNKAS WKGLQREQSS SEDTDAAFEL NSQLLPQENR LS LGHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Richter W, Hermsdorf T, Kronbach T, Dettmer D (2002) Protein Expression and Purification, 25, 138-148 |
| Project Aim | Folding |
| 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 |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pET21-C |
| Expression Protocol | 1L of LB medium was inoculated with 20ml overnight culture and incubated at 37degC until OD600 reached 0.6. IPTG was added to a final concentration of 0.1mM and incubation continued for a further 3hr. Cells were then harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | French Press |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 0.1M TrisHCl pH 7.0, 20mM EDTA |
| Solubilization Buffer | 6M GdnHCl, 0.1M TrisHCl pH 8.0 |
| Refolding Buffer | 50mM TrisHCl pH 8.5, 1M arginine hydrochloride, 50% ethylene glycol, 200mM MgCl2, 5mM DTT |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.5 |
| Refolding Temperature | 10.0 |
| Protein Concentration | |
| Refolding Time | 16h |
| Redox Agent | DTT |
| Redox Agent Concentration | 5mM |
| Refolding Protocol | Bacterial pellets (5g) were resupsended in 30mL 0.1M TrisHCl, pH 7.0, 1mM EDTA at 4degC. After addition of 7.5mg lysozyme, the solution was homogonized and incubated at 4degC for 30min. Cells were disrupted by 3 passages through a french press at 1200psig, then 10microg/ml DNase and 3mM MgCl2 were added followed by a further incubation at room temperature for 30min. After the addition of 0.5vol of 60mM EDTA, 6% Triton X-100, 1.5M NaCl pH 7.0, the suspension was incubated a further 30min at 4degC. Inclusion bodies were pelleted by centrifugation (30000g x 4degC x 10min), the pellet was resuspended in 40ml wash buffer and recentrifuged. This step was repeated 3 times. 80mg of inclusion bodies were solubilized at room temperature in solubilization buffer and centrifuged at 30000g for 10min. After determination of the protein conent, the inclusion bodies were added to refolding buffer at 10degC at a final concentration of 100microg/ml protein for 16h. 100ml of the renatured protein was diluted by stepwise addition of 100ml ddH20 and centrifuged at 4deg and 20000g for 10min. The batch was then concentrated by ultrafiltration to 20ml and then dialyzed overnight against 50mM TrisHCl, pH 7.5, 20% ethylene glycol. For purification using Zn-IDA-sepharose, the solution was then centrifuged (20000g x 4degC x 10min) and the supernatant applied to a 5ml Zn-IDA-sepharose column preoaded with 50mM ZnSO4 in sodium acetate buffer pH 4.0. and then equilibrated with 50ml of 50mM TrisHCl pH 7.5, 20% ethylene glycol. Following a wash with this buffer containing 25mM imidazole, the protein was eluted with the same buffer containing 150mM imidazole (1ml/min). Selected fractions were pooled and dialyzed against 50mM TrisHCl pH 7.5, 20% ethylene glycol. For purification using ResourceQ ion-exchange chromatography, the dialyzed protein was concentrated to about 10ml, then dialyzed again against 500ml of 50mM TrisHCl pH 8.5, 20% ethylene glycol, 2mM DTT at 4degC overnight. After centrifugation (30000g x 10min x 4degC), the supernatant was applied to a ResourceQ anion-exchange column at 1ml/min. After a wash, the prtoein was eluted with 50ml of a gradient from 0 to 200mM MgCl2 in 50mM TrisHCl pH 8.5, 20% ethylene glycol, 2mM DTT. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine,Ethylene glycol |
| Additives Concentration | 1M/50% |
| Refolding Yield | |
| Purity | |
| Notes | Activity yield using ResourceQ column better |