Refolding Record:
| Protein | |
|---|---|
| Protein Name | Ectonucleoside triphosphate diphosphohydrolase 6 (CD39L2) |
| Abbreviated Name | NTPDase6 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | O75354 |
| 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 | 485 |
| Molecular Weight | 53247.1 |
| Pi | 9.22 |
| Molecular Weight | 53247.1 |
| Disulphides | 2 |
| Full Sequence |
MKKGIRYETSRKTSYIFQQPQHGPWQTRMRKISNHGSLRVAKVAYPLGLCVGVFIYVAYIKWHRATATQAFFSITRAAPGARWGQQAHSPLGTAADGHEVFYGIMFDAGSTGTRVHVFQFTRPPRETPTLTHETFKALKPGLSAYADDVEKSAQGIRELLDVAKQDIPFDFWKATPLVLKATAGLRLLPGEKAQKLLQKVKEVFKASPFLVGDDCVSIMNGTDEGVSAWITINFLTGSLKTPGGSSVGMLDLGGGSTQIAFLPRVEGTLQASPPGYLTALRMFNRTYKLYSYSYLGLGLMSARLAILGGVEGQPAKDGKELVSPCLSPSFKGEWEHAEVTYRVSGQKAAASLHELCAARVSEVLQNRVHRTEEVKHVDFYAFSYYYDLAAGVGLIDAEKGGSLVVGDFEIAAKYVCRTLETQPQSSPFSCMDLTYVSLLLQEFGFPRSKVLKLTRKIDNVETSWALGAIFHYIDSLNRQKSPAS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Ivanenkov VV, Murphy-Piedmonte DM, Kirley TL. (2003) Biochemistry, 42, 117426-11735 |
| Project Aim | Expression and charactrization |
| Fusion | N-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 | 9 h |
| Expression Vector | pET28a |
| Expression Protocol | Expression of NTPDase6 in BL21(DE3) Cells. For bacterial expression, the NTPDase6 insert in the pET28a vector was transformed into E. coli BL21(DE3) cells. Five milliliters of LB broth containing 1% glucose and 60 g/mL kanamycin was inoculated with 10 L of frozen bacterial stock derived from a single colony of transformed BL21(DE3) cells and allowed to grow at 37 C for 5 h. This 5 mL culture was added to 500 mL of LB broth (containing 1% glucose and 30 g/mL kanamycin) and grown to an OD600 of approximately 0.5 before induction with IPTG (1 mM final concentration). After induction, the culture was grown for an additional 4 h at 37 C, reaching an OD600 of approximately 1. Bacterial inclusion bodies were prepared using the Pierce B-PER reagent as previously described (27), typically yielding 40-50 mg of inclusion body proteins. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M guanidine hydrochloride, 2 mM EDTA, and 5 mM DTT in 100 mM Tris-HCl, |
| Refolding Buffer | 250 mL of ice-cold buffer containing 100 mM Tris-HCl, 10 mM KCl, 250 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 500 mM (800 mM in some experiments) L-arginine hydrochloride, 2 mM reduced glutathione (GSH), and 0.4 mM oxidized glutathione (GSSG), pH 8.35 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.35 |
| Refolding Temperature | 23.0 |
| Protein Concentration | n/a |
| Refolding Time | 2-3 days |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2/0.4 mM |
| Refolding Protocol | Preparative Refolding of NTPDase6 from Inclusion Bodies. Approximately 5 mg of NTPDase6 inclusion body preparation was solubilized in 5 mL of 6 M guanidine hydrochloride, 2 mM EDTA, and 5 mM DTT in 100 mM Tris-HCl, denatured by heating for 10 min at 60 C, and then cooled to room temperature. Refolding was performed by dilution of the 5 mL sample into 250 mL of ice-cold buffer containing 100 mM Tris-HCl, 10 mM KCl, 250 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 500 mM (800 mM in some experiments) L-arginine hydrochloride, 2 mM reduced glutathione (GSH), and 0.4 mM oxidized glutathione (GSSG), pH 8.35, and proceeded for 2-3 days. Refolding experiments were performed at several different temperatures (ranging from 4 to 23 C) to determine the optimum temperature for refolding. After refolding, the 255 mL NTPDase6 sample was dialyzed against three 4 L changes of 50 mM Tris-HCl, 250 mM NaCl, and 2 mM CaCl2 (pH 8.0) for 2-3 days at 4 C to decrease the high arginine concentration that interferes with the hexahistidine tag binding to Ni-NTA beads. Purification of Refolded NTPDase6. The pET28a vector encodes a hexahistidine tag, N-terminal to the NTPDase6 insert, which allows purification of the protein on an immobilized metal affinity (Ni-NTA) column. Approximately 6 mL of Ni-NTA-agarose bead suspension (corresponding to a settled bead volume of approximately 3 mL) was washed three times with 10 mL of wash buffer containing 50 mM Tris-HCl, 250 mM NaCl, and 2 mM CaCl2, pH 8.0. Approximately 300 mL of refolded and dialyzed NTPDase6 was added to the Ni-NTA-agarose beads, and the slurry was incubated on ice for at least 1 h with gentle, occasional mixing. The NTPDase6/Ni-NTA-agarose slurry was poured into a column, and the unbound fraction was collected. The column was washed seven times with 4 mL of wash buffer. NTPDase6 was eluted from the Ni-NTA column in four 2.5 mL fractions of wash buffer containing 200 mM imidazole, pH 8.0. Eluted NTPDase6 was immediately diluted into 10 mL of ice-cold wash buffer (to a final sample volume of 20 mL) to decrease the imidazole concentration and promote protein solubility. All fractions, including the unbound material, were assayed for GDPase activity in the presence of Ca2+. |
| Refolding Assay | Circular Dichroism (uv-CD) |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 500 mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |