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| Refolding Record [Created: 2008-11-24 Updated: 2008-11-24 ] |
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PROTEIN |
| Protein Name |
CAMP-specific cyclic nucleotide phosphodiesterase PDE8A1 |
| Abbreviated Name |
PDE8A1 |
| SCOP Family |
Unknown |
| Structure Notes |
n/a |
| Organism |
Human |
| UniProt Accession |
Q96T71 |
| SCOP Unique ID |
n/a |
| Structure Solved |
homologue only |
| Class |
Unknown |
| Molecularity |
Unknown |
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CONSTRUCT |
| Full Length |
n |
| Domain |
catalytic domain (residues 480–820) |
| Chimera |
n/a |
| Variants |
n/a |
| Chain Length |
340 |
| Molecular Weight |
39287.4 |
| pI |
4.96 |
| Disulphides |
Unknown |
Pfam Domain(s) and
Number of Occurrences |
Pfam Entry
PDEase_I(1) |
| Domain Graphics |
 |
| Full Sequence |
LDDVPPRIARAMENEEYWDFDIFELEAATHNRPLIYLGLKMFARFGICEFLHCSESTLRSWLQIIEANYHSSNPYHNSTHSADVLHATAYFLSKERIKET
LDPIDEVAALIAATIHDVDHPGRTNSFLCNAGSELAILYNDTAVLESHHAALAFQLTTGDDKCNIFKNMERNDYRTLRQGIIDMVLATEMTKHFEHVNKF
VNSINKPLATLEENGETDKNQEVINTMLRTPENRTLIKRMLIKCADVSNPCRPLQYCIEWAARISEEYFSQTDEEKQQGLPVVMPVFDRNTCSIPKSQIS
FIDYFITDMFDAWDAFVDLPDLMQHLDNNFKYWKGLDEMK
BLAST against Refold database | BLAST against NCBI NR database
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| Notes |
n/a |
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EXPRESSION |
| Report |
Yan et al., ( 2008) Protein Expression and Purification, 1, 1 |
| Project Aim |
Protein refolding |
| Fusion |
N-terminal hexahistidine tag |
Protein Expression and
Production |
Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host |
E coli |
| Expression Strain |
BL21 |
| Expression Temperature |
37 |
| Expression Time |
overnight |
| Expression Vector |
pET15b-PDE8A1 |
| Expression Protocol |
The plasmid pET15b-PDE8A1 was transferred into E. coli strain BL21 (CodonPlus) for overexpression. The cell bearing the vector pET15b-PDE8A1 was grown in a modified 2xYT culture medium (16 g tryptone, 10 g yeast extract, and 5 g NaCl per liter) that was autoclaved before addition of 0.4% glucose, 100 mg ampicillin, and 20 mg chloramphenicol per liter. After the cell was grown at 37 °C to A600 = 0.7, 0.1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was added to induce the overexpression and the cell culture was transferred to room temperature for further growth overnight. |
| Method of Induction |
IPTG |
| Cell Density (at induction) |
OD 1 = 600 |
| Cell Disruption Method |
French Press |
| Lytic Agent |
None |
| Pre-refolding Purification |
Ni-NTA column |
| Solubility |
insoluble |
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REFOLDING |
| Refolding Method |
Dilution |
| Wash Buffer |
100 ml buffer of 8 M urea, 0.1 M Tris–HCl, pH 8.0, and eluted with the same buffer plus 0.5 M arginine |
| Solubilization Buffer |
6 M guanidine, 0.1 M Tris–HCl, pH 8.0 |
| Refolding Buffer |
0.5 M Tris–HCl, pH 7.0, 20 mM MgCl2, 20 mM MnCl2, 20 μM ZnSO4, 0.7 M arginine, 30% glycerol, 10 mM NaCl, 1 mM KCl, and 10 mM DTT |
| Pre-refolding Purification |
Ni-NTA column |
| Tag Cleaved |
no |
| Refolding pH |
7 |
| Refolding Temperature |
4 |
| Protein Concentration |
n/a |
| Refolding Time |
3 day |
| Redox Agent |
DTT |
| Redox Agent Concentration |
10 mM |
| Refolding Protocol |
About 10 g of the frozen cells from 2 l culture were suspended in 40 ml of the extraction buffer and passed through French Press three times at 1200 psi to disrupt them. The extraction buffer was 20 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM β-mercaptoethanol (β-ME), and 1 mM EDTA. The homogenate was centrifuged at 15,000 rpm in a Beckman JA-20 rotor for 30 min. fortunately, the recombinant PDE8A1 fragments mainly existed in the pellet phase and therefore refolding was necessary to obtain soluble and active PDE8A1 protein. The pellet was dissolved in 10 ml buffer of 6 M guanidine, 0.1 M Tris–HCl, pH 8.0, under slow orbital shaking at room temperature for 3 h. The dissolved mixture was centrifuged at 15,000 rpm for 20 min to remove the insoluble debris.The supernatant was loaded into a Ni–NTA column ( = 2.5 cm, 25 ml QIAGEN agarose beads). The column was washed with 100 ml buffer of 8 M urea, 0.1 M Tris–HCl, pH 8.0, and eluted with the same buffer plus 0.5 M arginine. The fractions of the PDE8A1 catalytic domain from the elution were combined. The protein concentration was estimated by the absorption of A280 (1.097 U = 1 mg/ml), as calculated by program ProtParam [38]. Fifty milligrams of the PDE8A catalytic domain at 2 mg/ml (25 ml of protein solution) was added dropwise into 1.7 l of the refolding buffer under mild stir. The refolding buffer was 0.5 M Tris–HCl, pH 7.0, 20 mM MgCl2, 20 mM MnCl2, 20 μM ZnSO4, 0.7 M arginine, 30% glycerol, 10 mM NaCl, 1 mM KCl, and 10 mM DTT. The refolding was carried out at 30 μg/ml protein concentration without shaking at 4 °C for 3 days.To concentrate the refolded PDE8A1, the dilute refolding system was mixed with 15 g of hydroxyapatite HTP GEL (Bio-Rad) that was presoaked in water. After stirred at room temperature for 15 min, the suspension was filtered with a filter paper. The beads were collected, re-suspended in 100 ml of 20 mM Tris–HCl, pH 8.0, 50 mM NaCl, 1 mM β-ME, and then packed into a column. The column was washed with 50 ml of the same buffer and eluted with 100 ml of 20 mM Tris–HCl, pH 8.0, 100 mM KH2PO4, 1 mM β-ME. The fractions were combined and dialyzed against 0.5 l of 20 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM β-ME twice, 1 h and overnight.To remove the His-tag, 2.5 mM CaCl2 and 1 μg/ml bovine thrombin (Haematologic Tech., Inc.) were added for digestion at 25 °C for 1 h. The digested PDE8A1 was loaded into a Q-Sepharose column (GE Healthcare) that was pre-equilibrated with a buffer of 50 mM NaCl, 20 mM Tris–HCl, pH 7.5, 1 mM β-ME, 1 mM MgCl2. The column was washed with 200 ml of 20 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mM β-ME, 1 mM EDTA, and then PDE8A1 was eluted out with the same buffer except for 300 mM NaCl. After being concentrated to about 10 ml, the protein was loaded into a gel filtration column Sephacryl S300 (GE Healthcare) and eluted with a buffer of 20 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM β-ME, 1 mM MgCl2. The protein was finally concentrated by Amicon cell and ultrafiltration membrane YM30 and its purity was estimated by the SDS gel.The fragment of PDE8A1 (205–820) that contains both PAS and catalytic domains was subcloned into pET15b and expressed in E. coli under the similar procedure, and refolded in a buffer of 50 mM Tris–HCl, pH 7.0, 1.25 M urea, 1 M arginine, 40 mM MnCl2, 10 mM NaCl, 1 mM KCl, and 10 mM DTT. |
| Assay |
enzyme activity |
| Chaperones |
None |
| Additives |
L-Arginine, Glycerol |
| Additives Concentration |
30%/0.7 M |
| Refolding Yield |
8 mg |
| Purity |
n/a |
| Notes |
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