Refolding Record:
| Protein | |
|---|---|
| Protein Name | Serotonin N-acetyltransferase |
| Abbreviated Name | AANAT |
| SCOP Family | N-acetyl transferase, NAT |
| Structure Notes | |
| Organism | Rat |
| UniProt Accession | Q64666 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 205 |
| Molecular Weight | 23141.9 |
| Pi | 7.64 |
| Molecular Weight | 23141.9 |
| Disulphides | Unknown |
| Full Sequence |
MLSIHPLKPEALHLPLGTSEFLGCQRRHTLPASEFRCLTPEDATSAFEIEREAFISVSGTCPLHLDEIRHFLTLCPELSLGWFEEGCLVAFIIGSLWDKERLTQESLTLHRPGGRTAHLHVLAVHRTFRQQGKGSVLLWRYLHHLGSQPAVRRAVLMCENALVPFYEKFGFQAMGPCAITMGSLTFTELQCSLRCHTFLRRNSGC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Ferry G, Ubeaud C, Dauly C, Mozo J, Guillard S, Berger S, Jimenez S, Scoul C, Leclerc G, Yous S, Delagrange P, Boutin JA. (2004) Protein Expression and Purification, 38, 84-98 |
| Project Aim | Purification & characterization |
| 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 | 24.0 |
| Expression Time | 6 h |
| Expression Vector | pGEX-4T |
| Expression Protocol | Expression, production, and purification of hAANAT have been described previously [14] and the same steps were used for ovine or rat AANAT. In brief, the arylalkylamine N-acetyltransferase cDNA coding regions, kindly provided by Dr. D.C. Klein and Dr. S.L. Coon (NIH, Bethesda, USA), was inserted into the bacterial expression vector pGEX-4T (Pharmacia, Les Ulis, France). An E. coli strain [BL21(DE3)pLysS] was transformed with the resulting plasmid. The expression of the construct was induced by adding isopropyl-1-thio-β-D-galactopyranoside (0.2 mM) at 24 °C for 6 h. The cells were harvested by centrifugation (5000g, 4 °C, 10 min). All the purification procedures were performed at 0–4 °C. Approximately 10 g of a frozen bacteria pellet was thawed in 40 ml of 2× PBS containing 10 mM dithiothreitol (DTT), a cocktail of protease inhibitors (Complete, Boehringer–Mannheim, 1 tablet per 50 ml), and the detergent Tween 85 (at 5% v/v). This suspension was sonicated eight times 1 min in ice, centrifuged (20,000g, 20 min), and the supernatant was slowly (40 ml/h) passed through an affinity column (8 mL glutathione–Sepharose, Pharmacia, Les Ulis, France), previously equilibrated with buffer A (2 × PBS, pH 6.9, containing 10 mM DTT). The column was washed (40 ml buffer A) and then eluted with 150 ml buffer B (50 mM Tris–HCl, pH 8.0, containing 100 nM sodium citrate, 10 mM DTT, and 10% (v/v) glycerol). The GST-AANAT was eluted sequentially with 40 ml of 10 mM glutathione in buffer B. Active fractions were pooled and stored frozen at −80 °C until use. The protein concentration was determined by the Bradford assay [26] (Protein Assay, BioRad, Marne-la-Coquette, France) with bovine serum albumin as standard. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Detergents |
| Pre-Refolding Purification | Ni-NTA column |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | 50 mM Tris–HCl, pH 8, containing 125 mM NaCl |
| Solubilization Buffer | 6 M guanidinium–HCl in buffer D (50 mM Tris–HCl, pH 8, containing 125 mM NaCl) |
| Refolding Buffer | on the column by changing the chaotropic agent from 6 M guanidium to 6 M urea |
| Pre-Refolding Purification | Ni-NTA column |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Denaturation, renaturation, and purification conditions of AANAT.V5-6His A pellet of transfected Tn5 was washed in 1% Triton X-100 in buffer D followed by centrifugation at 10,000g for 30 min at 4 °C. This process was repeated twice and washed again with buffer D without Triton. The last protein pellet was denatured by treatment with 6 M guanidinium–HCl in buffer D at room temperature and then applied on a 1.5 × 5 cm Ni–NTA column. The protein was refolded on the column by changing the chaotropic agent from 6 M guanidium to 6 M urea, after which the urea concentration was reduced from 6 to 0 M in 6 × 100 ml steps at a flow rate of 1 ml/min at room temperature. The column was then washed with 100 ml buffer D with 5 mM imidazole and the refolded protein was eluted in the same buffer with 100 mM EDTA. Protein was dialyzed against buffer C and stored in aliquots at −20 °C.Purification of AANAT.V5-6His with immobilized anti-v5 antibody affinity column The anti-V5 antibody was immobilized on protein G–Sepharose-4 fast flow (according to Amersham Bioscience protocol). The column was washed with 10 volumes of equilibrating buffer (100 mM phosphate, pH 7, containing 150 mM NaCl), followed by 10 volumes of buffer D to remove the excess of antibodies. Dialyzed Ni–NTA column fractions containing the hAANAT protein were combined and applied to this immunoaffinity column, which was further washed with buffer D. Elution was carried out with glycine buffer (pH 2.8) and the collected fractions were neutralized by 1 M Tris, pH 8. The eluted protein was dialyzed against buffer C and stored in aliquots at −20 °C. |
| Refolding Assay | enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |